Methods for preventing/treating damage to sensory hair cells and cochlear neurons

ABSTRACT

Methods for preventing or treating damage to sensory hair cells and cochlear neurons are disclosed. The methods comprise the administration of an effective amount of a compound of Formula I or Formula II. The method provides for the prevention/treatment of both hearing loss and loss of the sense of balance.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application Ser.No. 60/101,763, filed Sep. 25, 1998, the disclosure of which is herebyincorporated herein by reference, in its entirety.

TECHNICAL FIELD OF THE INVENTION

The present invention provides methods for preventing and/or treatinghearing loss and loss of the sense of balance. More specifically, thepresent invention provides methods for preserving sensory hair cells andcochlear neurons in a subject by administering an effective amount ofcompounds of Formula I and/or Formula II.

BACKGROUND OF THE INVENTION

The mammalian ear functions by transforming sound waves, or airbornevibrations, into electrical impulses. The brain then recognizes theseelectrical impulses as sound. The ear has three major parts, the outer,middle, and inner ear. Sound waves enter the outer ear and cause theeardrum to vibrate. The vibrations of the eardrum are transmittedserially through the three ossicles in the middle ear- the malleus,incus and stapes, also called the hammer, anvil and stirrup,respectively. The stirrup transmits the vibrations to the inner ear. Theinner ear comprises the cochlea and is connected to the middle ear viathe oval and round windows. The inner ear is filled with fluid andvibrations transmitted to the inner ear cause fluid movement in thecochlea of the inner ear. Fluid movement in the cochlea causes movementof sensory hair cells which initiates nerve impulses. These nerveimpulses are interpreted in the brain as sound.

The sensory hair cells are contained in the organ of Corti, which coilsaround the inside of the cochlea. Within the organ of Corti there areinner and outer sensory hair cells. The outer sensory hair cells arepresent in three rows, designated OHC1, OHC2 and OHC3; inner sensoryhair cells are present in one row. The sensory hair cells are attachedto the basilar membrane and contact the tectorial membrane. Movement offluids within the inner ear causes a movement of the basilar membranerelative to the tectorial membrane. This relative movement causes thecilia on the sensory hair cells to bend and leads to electricalactivity. Cochlear ganglion neurons below the sensory hair cellstransmit this electrical activity to auditory regions of the brain viathe auditory nerve.

The fluid filled inner ear, also called the membranous labyrinth,further contains the two mammalian organs of equilibrium which make upthe vestibular system. The first organ of equilibrium is composed of thesaccule and utricle which detect and convey information on body positionrelative to gravitational force. Both the saccule and utricle alsocontain sensory hair cells. Tiny particles of calcium carbonate lie onthe sensory hair cells in the saccule and utricle and bend the cilia tostimulate the sensory hair cells to send appropriate signals to thebrain, including “up”, “down”, “tilt” and “acceleration” in a particulardirection. Sensory hair cells in the utricle detect linear movement inthe horizontal plane while sensory hair cells in the saccule detectmovement in the vertical plane.

The second organ of equilibrium is composed of three semicircular canalswhich detect and convey information on movement, detected as fluidacceleration, to the brain. The semicircular canals are also lined withsensory hair cells, and are arranged at near 90 degree angles withrespect to one another and can detect movement in three dimensions. Asthe head is accelerated in one of these planes, fluid movement in thecanal corresponding to the plane of movement stimulates movement of thecilia of the sensory hair cells.

The vestibular organs—the saccule, the utricle and the semicircularcanals —stimulate nerve endings of vestibular ganglion neurons whichthen transmit information to a number of sites for different purposes.For example, information is transmitted from the vestibular system tothe eyes to keep the eyes focused on a target while the body is moving.Neurons also interconnect the vestibular system and the cerebellum forproducing smooth and coordinated bodily movements. Vestibularinformation also travels down the spinal cord to muscles in order tomaintain proper posture and balance.

Significant hearing loss causing communication problems occurs in aboutten percent of the population and more than one third of us will havesubstantial hearing loss by old age. Noise-induced hearing loss isestimated to be the cause of hearing loss in about one-third of the 28million Americans with hearing loss (NIH Publication No. 97-4233, April1997). In most cases, the auditory impairment results from the death ofsensory hair cells in the organ of Corti. Sensory hair cells aredelicate cells and thus are susceptible to damage from several sources,including, but not limited to, noise, infection, drugs, vascularinsufficiency and idiopathic effects. Idiopathic effects are thoseeffects which arise spontaneously or from an unknown or obscure cause.

Presbycusis is age-related hearing loss. Four distinct types ofpresbycusis have been described which are based upon audiograms andpathological analyses: 1) sensory—loss of sensory hair cells andsecondary degeneration of cochlear neuronal structures, 2) neural—lossof cochlear ganglion cells and/or nerve, 3) metabolic—atrophy of thestria vascularis, and 4) mechanical—stiffening of the basilar membrane(Schuknecht, Arch. Otol., 80:369-382, 1964). The neural and metaboliccauses of presbycusis may also result in the ultimate loss of haircells.

While no frequency data is associated with the descriptions of the typesof presbycusis, sensory presbycusis is the most common (Working Group onSpeech Understanding and Aging, Speech understanding and aging, J.Acoust. Soc. Am. 83:859-895, 1988). Johnsson et al. have described bothdegeneration of the stria vascularis and hair cell loss in 150 patientsranging in age from newborn to 97 years of age. Both are progressive andmost pronounced in elderly subjects. An age-related loss of hair cellsof the vestibular apparatus—saccule and utricle—was also noted that mayaccount for vestibular disturbances in the elderly (Johnsson et al.,Ann. Otol. Rhinol. Laryngol. 81:179-193, 1972; Johnsson et al., Ann.Otol. Rhinol. Laryngol. 81:364-376, 1972).

We are born with a complement of about 16,000 sensory hair cells and30,000 auditory neurons in each ear. These cells do not regenerateduring postnatal life. Therefore, loss of each cell, due to, forexample, noise, infection, toxic drugs (such as platinum-based cytotoxicagents and aminoglycosides) or idiopathic effects is irreversible andcumulative. If enough sensory cells are lost, the end result can betotal deafness.

Noise trauma is a widespread cause of hearing loss. Sound overexposurehas been demonstrated to lead to sensory hair cell apoptosis in theavian inner ear (Nakagawa et al., ORL, 59:303-310, 1997). There isincreasing evidence that the death of sensory hair cells caused by drugssuch as platinum-based cytotoxic agents and aminoglycosides ispartially, if not mainly, apoptotic. Noise-induced sensory hair cellloss in the cochlea apparently has a similar mechanism.

Aminoglycosides are widely used antibiotics used in patients withGram-negative bacterial infections (Paparella et al, Otolaryngology,1817, Saunders-Philadelphia, 1980). Aminoglycosides are known to causedamage to sensory hair cells and thereby affect hearing. Aminoglycosidesinclude, but are not limited to, neomycin, kanamycin, amikacin,streptomycin and gentamicin. Amikacin causes apoptosis of sensory haircells in rat cochleas (Vago et al., NeuroReport 9:431-436, 1998).Gentamicin treatment results in degeneration of sensory hair cells inguinea pigs (Li et al., J. Comparative Neur., 355:405-417, 1995; Lang etal., Hearing Res., 111:177-184, 1997).

The loss of sensory hair cells in the cochlea has been attributed toaminoglycoside ototoxicity. Apoptosis of sensory hair cells of guineapigs was observed following chronic treatment with aminoglycoside(Nakagawa et al., Eur. Arch. Otor., 254:9-14, 1997; Nakagawa et al.,Acta Otol., 255(3):127-131, 1998). Studies have assessed the protectiveeffect of various polypeptides on sensory hair cells in the cochlea.(See, for example, Malgrange et al., Abstr. Assoc. Res. Otol., 17:138,1994; Low et al., J. Cell. Physiol. 167:443-450, 1996; and Ernfors etal., Nature Medicine, 2:463-467, 1996). Ernfors et al. noted that,although the peptide NT-3 is a potent factor for preventing thedegeneration of spiral ganglion neurons, NT-3 “insufficiently protectsthe hair cells” (Ernfors et al., Nature Medicine, 2:463-467, 1996).

Platinum-based cytotoxic agents include, but are not limited to,cisplatin and carboplatin. Cisplatin is a widely used antitumor drugwhich causes structural changes in the inner ear and peripheral sensoryneuropathy. Hearing loss due to cisplatin is usually permanent andcumulative.

Rapid onset hearing loss, also named sudden sensorineural hearing loss,may also occur without any obvious reasons. Hearing loss in thesesituations develops either instantaneously or after a few hours. Thelocation of the damage is within the cochlea, and has been partiallyattributed to sensory hair cell damage. Such hearing loss may be due toidiopathic causes or as a result of other causes, including vasculardisease, hypertension and thyroid disease and viral infection by virusesincluding mumps, measles, mononucleosis, adenovirus, (Thurmond et al.,J. La. State Med. Soc., 150:201-203, 1998).

Damage to sensory hair cells and cochlear neurons may also occur as aresult of infection. For example, the onset of meningitis has beenlinked to hearing loss as a result of damage to sensory hair cells.(Blank et al., Arch. Otol. Head Neck Surg., 120:1342-1346, 1994).Meningitis as a result of E. Coli infection also damages sensory haircells (Marwick et al., Acta Otol. (Stockholm), 116(3):401-407, 1996).Toxins from Streptococcus pneumoniae have also been linked to damage tosensory hair cells (Comis et al., Acta Otol. (Stockholm) 113(2):152-159,1993).

Accessory epithelial structures of the cochlea and innervating cochlearneurons stay intact for a considerable length of time following trauma,but undergo secondary retrograde degeneration following the loss of IHCs(Ylikoski et al., 1974; Hawkins, 1976).

Several authors have recently shown that the cochlear sensory hair cellscan be protected to some extent from both ototoxic and noise damageusing various compounds. This was shown in animal model systems usinghair cell counts and hearing threshold measurements, e.g. by auditorybrainstem responses. The most commonly used therapeutic compounds havebeen antioxidants or free radical scavengers.

In addition to immediate mechanical damage, oxidative stress associatedwith the formation of free radicals (see discussion) and excitotoxicity(Basile et al., Nature Med. 2:1338-1343, 1996) have been implicated inthe pathogenesis of hearing loss. Evidence in various cell lines and invivo neuronal and non-neuronal model systems shows that apoptotic deathcan be induced by both oxidative stress and excitotoxicity (reviewed byPettmann and Henderson, Neuron 20:633-647, 1998). In the inner ear,necrotic hair cell death, characterized by cellular swelling, has beendemonstrated following acoustic trauma (Kellerhals, Adv. Oto-Rhino.Laryng. 18:91-168, 1972). More recent data, obtained in the ototoxicdrug-damaged inner ear, have suggested that hair cells may also diethrough apoptosis, based on the observations of nuclear fragmentation(Forge, Hear. Res. 19:171-182, 1985; Lee et al. J. Comp. Neur., vol.355,405-417, 1995;

Liu et al., Neuroreport 9:2609-2614, 1998; Nakagawa et al., Eur. Arch.

Otorhinolaryngol. 255:127-131, 1998; Vago et al., NeuroReport 9:431-436,1998). However, the contribution of apoptotic hair cell death to theloss of hearing function is not known. In addition, the molecularmechanisms involved in commitment to hair cell death are unknown.

Antioxidants and free radical scavengers have been tested because bothototoxic drug and noise damage have been postulated to produce an excessof reactive oxygen species (ROS) in the inner ear. Overproduction of ROSis thought to cause sensory hair cell damage by overwhelming thecochlea's antioxidant defense system (Ravi et al., Pharmacology andToxicology 76:386-394, 1995).

One of the signaling cascades that has been shown to mediate apoptoticdeath in response to a variety of stressful stimuli is thec-Jun-N-terminal kinase (JNK) pathway, also known as thestress-activated protein kinase (SAPK) pathway (Dérijard et al., Cell76:1025-1037, 1994; Kyriakis et al., Nature 369:156-160,1994). JNKactivation by phosphorylation has been shown to be important forneuronal cell death after trophic factor withdrawal in vitro and afterinjury in vivo (Xia et al., Science 270:1326-1331, 1995; Dickens et al.,Science 277:693-696, 1997; Yang et al., Nature 389:865-870, 1997). JNKsin turn phosphorylate c-Jun, a component of the transcription factorAP-1. Blockade of c-Jun activation and transcriptional activity in vitrohas been shown to prevent neuronal cell death (Estus et al., J. CellBiol. 127:1717-1727, 1994; Ham et al., Neuron 14:927-939, 1995; Watsonet al., J. Neurosci. 15:751-762, 1998). Recent data from c-Junphosphorylation-deficient mice (Behrens et al., Nature Gen. 21:326-329,1999) and from JNK knock-out mice (Yang et al., 1997) show that c-Junphosphorylation is essential for injury-induced neuronal death.

Neurotrophic factors including NT-3, BDNF and GDNF have also been shownto be important for protection of neurons within the inner ear, and mayalso have a role in hair cell protection after cochlear insult(Gabaizadeh et al., Acta Otol. (Stockholm), 117:232-235, 1997; Ernforset al. Ototoxicity: Basic Research and clinical applications, Savelletridi Fasano, Italy, Jun. 18-20, 1998, Abstract No. 12; Keithley et al.,Neuroreport, 9:(10), 2183-2187, 1988; Shoji et al., ARO Meeting, St.Petersburg Beach, Fla., Abstract No.539, 1998; Tay et al., ARO Meeting,St. Petersburg Beach, Fla., Abstract No.538, 1998; Ylikoski et al., HearRes 124:17-26, 1998). The loss of mechanoreception followingcisplatin-induced neuropathy has been reversed through theadministration of NT-3 (Gao et al., Ann. Neurol., 38:30-37, 1995).

Table 1, below, summarizes some compounds tested for protection ofcochlear sensory hair cells from damage, in vivo.

TABLE 1 TYPE OF TEST COMPOUND LESION REFERENCE antioxidants/free oxygenscavengers Lipid peroxidation noise Quirk et al., 1994¹ inhibitorR-phenylisopropanyl- noise Hu et al., 1997² adenosine Glutathionegentamicin Garetz et al., 1994³ Glutathione noise Yamasoba et al., 1998⁴D-methionine cisplatin Campbell et al, 1996⁵ D-methionine cisplatinGabaizadeh et al., 1997, (+BDNF) supra. Na-thiosulfate (STS) cisplatinKaltenbach et al., 1997⁶ neurotrotrophic factors BDNF cisplatinGabaizadeb et al., (+D-methionine) 1997, supra. NT-3 + MK801 noise,amikacin Emfors et al., 1998, supra. GDNF cisplatin Tay et al., 1998,supra. GDNF gentamicin Shoji et al., 1998, supra. GDNF noise Keithley etal., 1998, supra. others NMDA antagonists aminoglycosides Basile et al.,1996⁷ (MK801,ifenprodil) ORG 2766 (ACTH cisplatin DeGroot et al., 1997⁸analogue) Iron chelators gentamicin Song et al., 1997⁹ poly-1-asparticacid gentamicin Hulka et al., 1993¹⁰ Notated references from Table 1(not cited previously). ¹Quirk et al., Hear. Res., 74: 217-220, 1994.²Hu et al., Hear. Res., 113: 198-206, 1997. ³Garetz et al., Hear. Res.,77: 81-87, 1994. ⁴Yamasoba et al., Hear. Res., 784: 82-90, 1998.⁵Campbell et al., Hear. Res., 102: 90-98, 1996. ⁶Kaltenbach et al.,Otol. Head Neck Surg., 117: 493-500, 1997. ⁷Basile et al., Nat. Med., 2:1338-1343, 1996. ⁸DeGroot et al., Hear. Res., 106: 9-19, 1997. ⁹Song etal., J. Pharmacol. Exp. Therapeutics, 282: 369-377, 1997. ¹⁰Hulka etal., Am. J. Otol., 14: 352-356, 1993.

One problem in drug-based therapy of cochlear lesions is the limitedbiological activity of exogenously administered polypeptides. Thebiological half-life of many neurotrophic factors has been shown to bevery short. On the other hand, degeneration of sensory hair cells doesnot occur instantly; a large number of sensory hair cells at first seemto be reversibly damaged and might recover if treated promptly. Afternoise exposure, the typical pattern of cellular damage in the organ ofCorti takes 2-3 weeks to be complete. Affected cochlear neurons start todegenerate after noise has destroyed the sensory hair cells and thenerve terminals, 3-4 weeks postexposure.

There is no effective medical treatment to date for auditory sensoryhair cell loss. Also, prevention of sensory hair cell degeneration isobscured by the fact that exact molecular mechanisms of damage to theauditory organ are unknown. Consequently, no effective regimen has beendeveloped to prevent or treat damage to sensory hair cells. Therefore,there exists a need for compositions and methods to prevent and/or treatsensory hair cell damage.

There is also no effective medical treatment known to date for loss ofcochlear neurons. Therefore, a need exists for compositions and methodsto prevent and/or treat damage or loss of cochlear neurons.

As is clear from the foregoing discussion, damage to sensory hair cellsor cochlear neurons can also affect the vestibular system and canresult, for example, in vertigo. Benign paroxysmal positional vertigo(BPPV) affects about 40 to 60 people per 100,000 population every year.Also, Meniere's disease affects about 40 people per 100,000 populationeach year. During the course of these and other diseases, the sensoryhair cells of the vestibular system have a tendency to degenerate. Noeffective regimen to date has been developed to prevent or treat damageto sensory hair cells in the vestibular system. Therefore, there existsa need for compositions and methods to prevent and/or treat damage tosensory hair cells and neurons in the vestibular system.

SUMMARY OF THE INVENTION

According to one aspect of the invention, a method is provided forpreventing hearing loss in a subject comprising administering to saidsubject an effective amount of a fused pyrrolocarbazole of Formula Ihaving the formula:

or a stereoisomer or pharmaceutically acceptable salt form thereof,wherein:

ring D is selected from phenyl and cyclohexene with double bond a-b;

ring B and ring F are independently selected from:

(a) a 6-membered carbocyclic ring in which from 1 to 3 carbon atoms maybe replaced by heteroatoms;

(b) a 5-membered carbocyclic ring; and

(c) a 5-membered carbocyclic ring in which either:

(1) one carbon atom is replaced with an oxygen, nitrogen, or sulfuratom;

(2) two carbon atoms are replaced with a sulfur and a nitrogen atom, anoxygen and a nitrogen atom, or two nitrogen atoms; or

(3) three carbon atoms are replaced with three nitrogen atoms, oneoxygen and two nitrogen atoms, or one sulfur and two nitrogen atoms;

G-X-W is selected from:

(a) (Z¹Z²)C—N(R¹)—C(Z¹Z²);

(b) CH(R¹)—C(=O)—N(R¹); and

(c) N(R¹)—C(=O)—CH(R¹);

Z¹ and Z², at each occurrence, are independently selected from H, H; H,OR; H, SR; H, N(R)₂; and a group wherein Z¹and Z² together form a moietyselected from =O, =S, and =NR; with the proviso that at least one of thepairs Z¹ and Z² forms =O;

R is independently selected from H, optionally substituted alkyl, OH,alkoxy, OC(=O)R^(1a), OC(=O)NR^(1c)R^(1d), O(CH₂)_(p)NR^(1c)R^(1d),O(CH₂)_(p)OR^(1b), optionally substituted arylalkyl and optionallysubstituted heteroarylalkyl;

R¹ is independently selected from:

(a) H, optionally substituted alkyl, optionally substituted aryl,optionally substituted arylalkyl, optionally substituted heteroaryl andoptionally substituted heteroarylalkyl;

(b) C(=O)R^(1a);

(c) OR^(1b);

(d) C(=O)NHR^(1b), NR^(1c)R^(1d), (CH₂)_(p)NR^(1c)R^(1d),(CH₂)_(p)OR^(1b), O(CH₂)_(p)OR^(1b) and O(CH₂)_(p)NR^(1c)R^(1d),

R^(1a) is independently selected from optionally substituted alkyl,optionally substituted aryl and optionally substituted heteroaryl;

R^(1b) is independently selected from H and optionally substitutedalkyl;

R^(1c) and R^(1d) are each independently selected from H, optionallysubstituted alkyl and a linking group of the formula (CH₂)₂—X¹—(CH₂)₂;

X¹ is independently selected from O, S and CH₂;

Q is selected from NR², 0, S, NR²², CHR²³, X⁴CH(R²³), CH(R²³)X⁴, whereinX⁴ is selected from O, S, CH₂, NR²² and NR^(2;)

R² is selected from H, SO₂R^(2a), CO₂R^(2a), C(=O)R^(2a),C(=O)NR^(2c)R^(2d), optionally substituted alkyl, optionally substitutedalkenyl and optionally substituted alkynyl, wherein said optionalsubstituents are one to about three R⁵ groups;

R^(2a) is independently selected from optionally substituted alkyl,optionally substituted aryl, optionally substituted carbocyclyl,optionally substituted heterocyclyl, OR^(2b), CONH₂, NR^(2c)R^(2d),(CH₂)_(p)NR^(2c)R^(2d) and O(CH₂)_(p)NR^(2c)R^(2d);

R^(2b) is selected from H and optionally substituted alkyl;

R^(2c) and R^(2d) are each independently selected from H and optionallysubstituted alkyl, or together form a linking group of the formula(CH₂)₂—X¹—(CH₂)₂;

R³ and R⁴ are each independently selected from:

(a) H, aryl, heteroaryl, F, Cl, Br, I, CN, CF₃, NO₂, OH, OR⁹,O(CH₂)_(p)NR¹¹R¹², OC(=O)R⁹, OC(=O)NR¹¹R¹², O(CH₂)_(p)OR¹⁰, CH₂OR¹⁰,NR¹¹R¹², NR¹⁰S(=O)₂R⁹ and NR¹⁰C(=O)R⁹;

(b) CH₂OR¹⁴;

(c) NR¹⁰C(=O)NR¹¹R¹², CO₂R¹⁰, C(=O)R⁹, C(=O)NR¹¹R¹², CH=NOR¹⁰, CH=NR¹⁰,(CH₂)_(p)NR¹¹R¹², (CH₂)_(p)NHR¹⁴ and CH=NNR¹¹R¹²;

(d) S(O)_(y)R⁹, (CH₂)_(p)S(O)_(y)R⁹ and CH₂S(O)_(y)R¹⁴;

(e) optionally substituted alkyl, optionally substituted alkenyl andoptionally substituted alkynyl, wherein said optional substituents areone to about three R⁵ groups;

R⁹ is selected from alkyl, (CH₂)_(r)aryl and (CH₂)_(r)heteroaryl;

R¹⁰ is selected from H, alkyl, (CH₂)_(r)aryl and (CH₂)_(r)heteroaryl;

R¹¹ and R¹² are independently selected from H and optionally substitutedalkyl, or together form a linking group of the formula (CH₂)₂—X¹—(CH₂)₂;

R⁵ is selected from aryl, heteroaryl, arylalkoxy, heterocycloalkoxy,hydroxyalkoxy, alkyloxy-alkoxy, hydroxyalkylthio, alkoxy-alkylthio, F,Cl, Br, I, CN, NO₂, OH, OR⁹, X²(CH₂)_(p)NR¹¹R¹²,X²(CH₂)_(p)C(=O)NR¹¹R¹², X²(CH₂)_(p)OC(=O)NR¹¹R¹², X²(CH₂)_(p)CO₂R⁹,X²(CH₂)_(p)S(O)_(y)R⁹, X²(CH₂)_(p)NR¹⁰C(=O)NR¹¹R¹², OC(=O)R⁹,OC(=O)NHR¹⁰, O-tetrahydropyranyl, NR¹¹R¹², NR¹⁰C(=O)R⁹, NR¹⁰CO₂R⁹,NR¹⁰C(=O)NR¹¹R¹², NHC(=NH)NH₂, NR¹⁰S(O)₂R⁹, S(O)_(y)R⁹, CO₂R¹⁰,C(=O)NR¹¹R¹², C(=O)R⁹, CH₂OR¹⁰, CH=NNR¹¹R¹², CH=NOR¹⁰, CH=NR⁹,CH=NNHCH(N=NH)NH₂, S(=O)₂NR¹¹R¹², P(=O)(OR¹⁰)₂, OR¹⁴, and amonosaccharide wherein each hydroxyl group of the monosaccharide isindependently either unsubstituted or is replaced by H, alkyl,alkylcarbonyloxy, or alkoxy;

X² is O, S, or NR¹⁰;

Y is selected from:

(a) a direct bond;

(b) optionally substituted CH₂, CH₂CH₂ or CH₂CH₂CH₂, wherein saidoptional substituents are one to about three R¹⁹ groups; and

(c) CH=CH, CH(OH)—CH(OH), O, S, S(=O), S(=O)₂, C(R¹⁸)₂, C=C(R¹⁹)₂,C(=O), C(=NOR²⁰), C(OR²⁰)R²⁰, C(=O)CH(R¹⁸), CH(R¹⁸)C(=O),C(=NOR²⁰)CH(R¹⁸), CHR²¹C(=NOR²⁰), C(=O)N(R²¹), N(R²¹)C(=O), CH₂Z, ZCH₂and CH₂ZCH₂, where Z is selected from C(R²⁰)₂, O, S, CO₂R²⁰, C(=NOR²⁰)and N(R²⁰);

R¹⁸ is independently selected from H, SO₂R^(18a), CO₂R^(18a),C(=O)R^(18a), C(=O)NR^(18c)R^(18d), optionally substituted alkyl,optionally substituted alkenyl, and optionally substituted alkynyl;

R^(18a) is independently selected from optionally substituted alkyl,optionally substituted aryl, optionally substituted carbocyclyl andoptionally substituted heterocyclyl;

R^(18c) and R^(18d) are each independently selected from H andoptionally substituted alkyl, or together form a linking group of theformula (CH₂)₂—X¹—(CH₂)₂;

R¹⁹ is independently selected from R²⁰, thioalkyl, halogen, optionallysubstituted alkyl, optionally substituted alkenyl and optionallysubstituted alkynyl;

R²⁰ is independently selected from H, alkyl, OH, alkoxy, OC(=O)R^(18a),OC(=O)NR^(18c)R^(18d), OC(=S)NR^(18c)R^(18d), O(CH₂)_(p)NR^(18c)R^(18d),O(CH₂)_(p)OR²¹, optionally substituted arylalkyl, optionally substitutedheterocyclylalkyl and optionally substituted carbocyclyl;

R²¹ is independently selected from H and alkyl;

Q′ is selected from:

(a) a direct bond;

(b) NR^(6;)

(c) optionally substituted CH₂, CH₂CH₂ or CH₂CH₂CH₂;

(d) CR²²R²⁴; and

(e) CH=CH, CH(OH)CH(OH), O, S, S(=O), S(=O)₂, C(=O), C(=NOR¹¹),C(OR¹¹)(R¹²), C(=O)CH(R¹³), CH(R¹³)C(=O), C(R¹⁰)₂, C(=NOR¹¹)CH(R¹³),CH(R¹³)C(=NOR¹¹), CH₂Z′, Z′—CH₂ and CH₂Z′CH₂;

Z′ is selected from C(R¹¹)(OR¹²), O, S, C(=O), C(=NOR¹¹) and NR¹¹;

R⁶ is selected from H, SO₂R^(2a), CO₂R^(2c) C(=O)R^(2a),C(=O)NR^(1c)R^(1d) , optionally substituted alkyl, optionallysubstituted alkenyl, and optionally substituted alkynyl, wherein saidoptional substituents are one to about three R⁵ groups; or

alternatively, when Q is NR² and Q′ is NR⁶ or C(R¹⁰)₂, R² and R⁶ or oneof R¹⁰ are joined together to form:

wherein R⁷ and R⁸ are each independently selected from H, OH, alkyl,alkoxy, optionally substituted arylalkyl, optionally substitutedheteroarylalkyl, (CH₂)_(p)OR¹⁰, (CH₂)_(p)OC(=O)NR¹¹R¹² and(CH₂)_(p)NR¹¹R¹²; or R⁷ and R⁸ together form a linking group of theformula CH₂—X³—CH₂;

X³ is a bond, O, S, or NR¹⁰;

J is selected from a bond, O, CH=CH, S, C(=O), CH(OR¹⁰), N(OR¹⁰),N(OR¹⁰), CH(NR¹¹R¹²), C(=O)N(R¹⁷), N(R¹⁷)C(=O), N(S(O)_(y)R⁹),N(S(O)_(y)NR¹¹R¹²), N(C(=O)R¹⁷), C(R¹⁵R¹⁶), N⁺(O⁻)(R¹⁰), CH(OH)CH(OH)and CH(O(C=O)R⁹)CH(OC(=O)R⁹);

J′ is selected from O, S, N(R¹⁰), N⁺(O⁻)(R¹⁰), N(OR¹⁰) and CH₂;

R¹³ is selected from alkyl, aryl and arylalkyl;

R¹⁴ is the residue of an amino acid after the hydroxyl group of thecarboxyl group is removed;

R¹⁵ and R¹⁶ are independently selected from H, OH, C(=O)R¹⁰, O(C=O)R⁹,alkyl-OH, alkoxy and CO₂R¹⁰;

R¹⁷ is selected from H, alkyl, aryl and heteroaryl;

R²²is

X⁵ and X⁶ are independently selected from O, N, S, CHR²⁶, C(OH)R²⁶,C(=O) and CH₂=C;

X⁷ and X⁸ are independently selected from a bond, O, N, S, CHR²⁶,C(OH)R²⁶, C(=O) and CH₂=C;

X⁹ and X¹⁰ are independently selected from a bond, O, N, S, C(=O) andCHR²⁶;

X¹¹ is a bond or alkylene optionally substituted with NR¹¹R¹² or OR³⁰;

R²³ is selected from H, OR²⁷, SR²⁷, R²² and R²⁸;

R²⁴ is selected from R, thioalkyl, and halogen;

R²⁵ is selected from R¹ and OC(=O)NR^(1c)R^(1d);

R²⁶ is selected from H, optionally substituted alkyl and optionallysubstituted alkoxy, wherein

(1) ring G contains 0 to about 3 ring heteroatoms;

(2) any two adjacent hydroxyl groups of ring G can be joined to form adioxolane ring;

(3) any two adjacent ring carbon atoms of ring G can be joined to form afused aryl or heteroaryl ring; with the provisos that:

(a) when X¹¹ is a bond, ring G can be heteroaryl; and

(b) ring G:

(i) contains at least one carbon atom that is saturated;

(ii) does not contain two adjacent ring O atoms;

(iii) contains a maximum of two C(=O) groups;

R²⁷ is selected from H and alkyl;

R²⁸ is selected from aryl, arylalkyl, SO₂R²⁹, CO₂R²⁹, C(=O)R²⁹,optionally substituted alkyl, optionally substituted alkenyl andoptionally substituted alkynyl;

R²⁹ is selected from alkyl, aryl and heteroaryl;

R³⁰ is selected from H, alkyl, acyl and C(=O)NR¹¹R¹²;

m is independently selected from 0, 1, and 2;

p is independently selected from 1, 2, 3, and 4;

r is independently selected from 0, 1, and 2;

y is independently selected from 0, 1 and 2; and

z is selected from 0, 1, 2, 3 and 4;

with the provisos that at least one of Y and Q′ is a direct bond, when Yis a direct bond, Q′ is other than a direct bond, when Q′ is a directbond, Y is other than a direct bond, and when rings B and F are phenyl,G-X-W is CH₂NHC(=O), Y is a direct bond, Q is NR² and Q′ is NR⁶ where R⁶is joined with R² to form

then R³ is other than CH₂SCH₂CH₃.

Another aspect of the invention provides a method for preventing loss ofsense of balance in a subject comprising administering to said subjectan effective amount of a fused pyrrolocarbazole of Formula I, as definedabove.

Yet another aspect of the invention provides a method for preventing thedeath of sensory hair cells in a subject comprising administering aneffective amount of the compound of Formula I, as defined above.

A further aspect of the invention provides a method for preventingsudden sensorineural hearing loss due to the loss of sensory hair cellscomprising administering an effective amount of the compound of FormulaI, as defined above.

Another aspect of the invention provides a method for preservingfunction of sensory hair cells prior to or subsequent to trauma in asubject comprising administering an effective amount of the compound ofFormula I, as defined above.

Yet another aspect of the invention provides a method for treatingdamaged sensory hair cells comprising administering an effective amountof the compound of Formula I, as defined above.

A further aspect of the invention provides a method for preventing deathof cochlear neurons in a subject comprising administering an effectiveamount of Formula I, as defined above.

A further aspect of the present invention provides a method forpreventing hearing loss in a subject comprising administering to saidsubject an effective amount of the compound of Formula II;

A further aspect of the present invention provides a method forpreventing loss of sense of balance in a subject comprisingadministering to said subject an effective amount of the compound ofFormula II as defined above.

A still further aspect of the present invention provides a method forpreventing death of sensory hair cells in a subject comprisingadministering to said subject an effective amount of the compound ofFormula II as defined above.

A further aspect of the present invention provides a method forpreventing sudden sensorineural hearing loss in a subject due to deathof sensory hair cells comprising administering to said subject aneffective amount of the compound of Formula II as defined above.

A further aspect of the present invention provides a method forpreserving function of sensory hair cells prior to or subsequent totrauma in a subject comprising administering to said subject aneffective amount of the compound of Formula II as defined above.

A still further aspect of the present invention provides a method forpreventing death of cochlear neurons in a subject comprisingadministering to said subject an effective amount of the compound ofFormula II as defined above.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts ABR (auditory brainstem response) measurements anddemonstrates that the compound of Formula I prevents noise-inducedsensory hair cell loss. ABR measurements were made at 2, 4, 8, 16 and 32kHz prior to noise-induced lesion (filled diamond). Four animals weregiven the compound of Formula II (1 mg/kg, s.c.) followed by noisetrauma 24 hours later. Animals were maintained for 21 days; treatedanimals were dosed once per day with the compound of Formula II at 1mg/kg. ABR measurements were again taken at the end of 21 days. Lesioncontrols are shown as filled triangles; the compound of Formula IItreated animals are shown as filled squares.

FIGS. 2a and 2 b depict treatment with the compound of Formula II.Treatment with the compound of Formula II preserved outer sensory haircells throughout the organ of Corti. Cells were counted from one end ofthe organ in millimeters (X-axis) and the number of cells remainingafter treatment are represented as a percentage of the total numberoriginally present (sum of cells plus Deiters scars) on the Y-axis. FIG.2a shows the number of OHCs in animals treated with the compound ofFormula II, subject identification numbers (SIN) 13, 14, 19 and 20. FIG.2b shows the number of OHCs present in control animals, SIN 15, 17 and18. Three out of four compound of Formula II treated animals (SIN 13, 14and 19) showed only a minor loss of sensory hair cells ranging from 1-3%(Table 2). The fourth animal (SIN 20) had a loss of 19%. In the threeanimals with almost complete sensory hair cell preservation, the smallpercentage of cells lost did not appear to localize to a particular sitein the organ of Corti (FIG. 2a).

FIGS. 3a-f depict hair cell death in the guinea pig cochlea one dayafter noise exposure (120 dB, 4 kHz, 6 hr). The transverse (midmodiolar)paraffin section of the organ of Corti in the second cochlear turn isdouble stained with DAPI nuclear stain (FIG. 3a) and with TUNEL-method(FIG. 3b). The 3 outer hair cells (arrow) show distorted nuclei and areTUNEL-positive. Supporting cells, seen below the hair cells, are notTUNEL-labeled. FIG. 3c shows a TUNEL-labeled paraffin section and showsan outer hair cell nucleus with DNA fragmentation at high magnification.FIG. 3d depicts a toluidine blue-stained, resin-embedded semi-thinsection of the organ of Corti in transverse plane and shows an innerhair cell (large arrow) and 3 rows of outer hair cells (OHCs, smallarrows). Only OHC1 shows a fragmented nucleus (thick arrow). The sectionshown is from the area of scattered hair cell loss. (De=Deiter's cells.)FIG. 3e shows a toluidine blue-stained, resin-embedded semi-thin sectionin horizontal plane and at the level of hair cell nuclei. The section isfrom the region of maximal trauma. Most hair cells are lost, except oneouter hair cell that shows a fragmented nucleus (thick arrow). FIG. 3fdepicts a section, prepared as in FIG. 3e, from a nontraumatized regionof the organ of Corti. Outer hair cells of the 3 rows are present andtheir nuclei are not fragmented. Scale bar represents 80 mm in FIGS. 3aand 3 b, 10 mm in FIG. 3c, 18 mm in FIG. 3d, and 15 mm in FIGS. 3e and 3f.

FIGS. 4a-c depict the protection against permanent hearing loss aftertreatment with the compound of compound of Formula II measured by ABR.FIG. 4a shows ABR threshold shifts 2 days after noise trauma. FIG. 4bshows ABR threshold shifts 6 days after noise trauma. FIG. 4c shows ABRshows ABR threshold shifts 14 days after noise trauma. Results showmean±SEM. Six and 14 days after exposure, the average threshold shiftsof control animals are significantly greater than that of FormulaII-treated animals. (***P<0.001; **P<0.05; Student's t-test.)

FIGS. 5a and 5 b depict the prevention of loss of sensory hair cells twoweeks after traumatic noise exposure in animals treated with thecompound of Formula II. Cochleograms of control (FIG. 5a, n=13) andFormula II-treated animals (FIG. 5b, n=12) revealed that a significantlylower percentage of sensory hair cells were lost in treated animalsversus control animals. Results show mean±SEM.

FIGS. 6a and 6 b depict the preservation of sensory hair cells followingneomycin treatment due to treatment with the compound of Formula II.Organ of Corti explants of neonatal rats were dissected for surfacepreparations, treated with 100 μM neomycin alone or in conjunction withthe compound of Formula II (500 nM) for 48 hours, stained withphalloidin, and imaged by laser confocal microscope (FIG. 6a). Neomycincauses degeneration of a large number of hair cells (FIG. 6 b). Thecompound of Formula II protects hair cells from neomycin induced celldeath.

FIGS. 7a-c depicts the prevention of hair cell loss in cochlear explantsof neonatal rats exposed to neomycin for 48 hr. As shown inphalloidin-labeled confocal images, FIG. 7a shows the loss of hair cellscaused by Neomycin. FIG. 7b shows the protective effect of Formula II inpreventing hair cell loss in the basal turn of the cochlea. (i=innerhair cell; 1,2,3=rows of outer hair cells), and FIG. 7c shows the numberof protected outer hair cells in basal and middle turns. Histograms andbars represent mean ± SEM for 3 experiments, each including 4 explantsof both the control and Formula II-treated groups. The scale barrepresents 50 mm in FIGS. 7a and 7 b.

FIG. 8 shows the attenuation of gentamicin-induced hearing loss throughadministration of Formula II. Formula II treated cochleas showedstatistically significantly less threshold shift at all frequenciestested than control cochleas.

FIG. 9 shows the effects of Formula II (500 nM), neurotrophin-3 (2ng/ml) and nerve growth factor (2 ng/ml) on the survival of neonatalcochlear neurons in vitro. Numbers of neurons in cultures with addedcompounds are expressed as percentage of the number of neurons incontrol cultures. Neurons were counted after 48 hr in culture. Valuesrepresent mean ± SEM from 3 separate experiments. Formula II was asefficacious as NT-3 in promoting survival of cochlear neurons. NGFserved as a negative control.

FIG. 10 depicts the effect of delayed dosing of Formula II followingnoise-induced hearing loss. Although attenuation of hearing loss is mosteffective following administration of Formula II prior to the noiselesion, administration of Formula II both 1 and 4 days subsequent tonoise lesioning attenuates hearing loss compared to control.

FIGS. 11a-d depict the effect of delayed dosing of Formula II followingnoise-induced hearing loss. Cochleograms of Formula II-treated animals(FIGS. 11a-c, n=12) and control (FIG. 11d, n=13) revealed that asignificantly lower percentage of sensory hair cells were lost intreated animals versus control animals.

Results show mean ± SEM.

FIGS. 12a-f depict hair cell death, and JNK and c-Jun phosphorylation inneomycin-exposed (100 mM) cochlear explants of neonatal rats. Thespecimens were embedded in paraffin and cut in transverse (midmodiolar)plane. FIG. 12a shows one row of calbindin-immunoreactive inner haircells and 3 rows of outer hair cells (arrows) are seen in nonexposedexplants. FIG. 12b shows TUNEL-stained outer hair cell nuclei (arrows)are seen in cultures exposed to neomycin for 12 hr. FIG. 12c showshigher magnification of an outer hair cell nucleus showingTUNEL-positive DNA fragmentation. FIGS. 12d and 12 e show thatphospho-JNK and phospho-c-Jun immunolabeling, respectively, is found inthe nuclei of hair cells (arrows) exposed to neomycin for 6 hr. FIG. 12fshows that phospho-c-Jun immunoreactive hair cells are not seen incultures coincubated with neomycin and Formula II (500 nM) for 6 hr.Arrows point to hair cells. Scale bar represents 27 mm in FIGS. 12a, 12b, 12 d, 12 e, and 12 f, 10 mm in FIG. 12c.

DETAILED DESCRIPTION OF THE INVENTION

As employed above and throughout our disclosure, the following terms,unless otherwise indicated, shall be understood to have the followingmeanings.

As used herein, the term “alkyl” means a straight-chain, cyclic, orbranched alkyl group having 1 to 8 carbon atoms, such as methyl, ethyl,propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl,isoamyl, neopentyl, 1-ethylpropyl, hexyl, octyl, cyclopropyl, andcyclopentyl. The alkyl moiety of alkyl-containing groups, such asalkoxy, alkoxycarbonyl, and alkylaminocarbonyl groups, has the samemeaning as alkyl defined above. Lower alkyl groups, which are preferred,are alkyl groups as defined above which contain 1 to 4 carbons.

Alkyl groups and alkyl moieties contained within substituent groups suchas aralkyl, alkoxy, arylalkoxy, hydroxyalkoxy, alkoxy-alkoxy,hydroxy-alkylthio, alkoxy-alkylthio, alkylcarbonyloxy, hydroxyalkyl andacyloxy groups may be substituted or unsubstituted. A substituted alkylgroup has 1 to 3 independently-selected substituents, preferablyhydroxy, lower alkoxy, lower alkoxy-alkoxy, substituted or unsubstitutedarylalkoxy-lower alkoxy, substituted or unsubstitutedheteroarylalkoxy-lower alkoxy, substituted or unsubstituted arylalkoxy,substituted or unsubstituted heterocycloalkoxy, halogen, carboxyl, loweralkoxycarbonyl, nitro, amino, mono- or di-lower alkylamino, dioxolane,dioxane, dithiolane, dithione, furan, lactone, or lactam.

As used herein, the term “alkenyl” is intended to includestraight-chain, cyclic, or branched hydrocarbon chains having at leastone carbon-carbon double bond. Examples of alkenyl groups includeethenyl, propenyl, 3-methylbutenyl, and cyclohexenyl groups. As usedherein, the term “alkynyl” is intended to include straight-chain,cyclic, or branched hydrocarbon chains having at least one carbon-carbontriple bond. Examples of alkynyl groups include ethynyl, propynyl,3-methylbutynyl, and cyclohexynyl groups.

As used herein, the “acyl” moiety of acyl-containing groups such asacyloxy groups is intended to include a straight-chain, branched, orcyclic alkanoyl group having 1 to 6 carbon atoms, such as formyl,acetyl, propanoyl, butyryl, valeryl, pivaloyl or hexanoyl.

As used herein, the term “carbocyclic” or “carbocyclyl” refers to cyclicgroups in which the ring portion is composed solely of carbon atoms.These include, but are not limited to, cyclopropyl, cyclobutyl,cyclopentyl, cyclohexl, cycloheptyl, cyclooctyl. The term “carbocyclicaromatic ring” is intended to include carbocyclic rings which are alsoaryl rings. The terms “heterocyclo”, “heterocyclic” and “heterocyclyl”refer to cyclic groups in which the ring portion includes at least oneheteroatom such as O, N, or S. Heterocyclyl groups include heteroaryland heteroalkyl groups.

As used herein the term “aryl” means an aromatic ring having 6 to 12carbon atoms such as phenyl, biphenyl and naphthyl. Preferred arylgroups include unsubstituted or substituted phenyl and naphthyl groups.The term “heteroaryl” as used herein denotes an aryl group in which oneor more ring carbon atoms is replaced by a hetero (i.e., non-carbon)atom such as O, N or S. Preferred heteroaryl groups include pyridyl,pyrimidyl, pyrrolyl, furyl, thienyl, imidazolyl, triazolyl, tetrazolyl,quinolyl, isoquinolyl, benzoimidazolyl, thiazolyl, pyrazolyl, andbenzothiazolyl groups. The term “heteroalkyl” denotes a cycloalkyl groupin which one or more ring carbon atoms is replaced by hetero atoms suchas O, N, or S.

As used herein, the term “aralkyl” (or “arylalkyl”) is intended todenote a group having from 7 to 15 carbons, consisting of an alkyl groupthat bears an aryl group. Examples of aralkyl groups include, but arenot limited to, benzyl, phenethyl, benzhydryl and naphthylmethyl groups.Substituted aryl, substituted heterocyclic and substituted aralkylgroups each have 1 to 3 independently selected substituents that arepreferably lower alkyl, hydroxy, lower alkoxy, carboxy, loweralkoxycarbonyl, nitro, amino, mono- or di-lower alkylamino, and halogen.

Preferred heterocyclic groups formed with a nitrogen atom includepyrrolidinyl, piperidinyl, piperidino, morpholinyl, morpholino,thiomorpholino, N-methylpiperazinyl, indolyl, isoindolyl, imidazole,imidazoline, oxazoline, oxazole, triazole, thiazoline, thiazole,isothiazole, thiadiazoles, triazines, isoxazole, oxindole, indoxyl,pyrazole, pyrazolone, pyrimidine, pyrazine, quinoline, iosquinoline, andtetrazole groups. Preferred heterocyclic groups formed with an oxygenatom include furan, tetrahydrofuran, pyran, benzofurans, isobenzofurans,and tetrahydropyran groups. Preferred heterocyclic groups formed with asulfur atom include thiophene, thianaphthene, tetrahydrothiophene,tetrahydrothiapyran, and benzothiophenes.

As used herein, “hydroxyalkyl” groups are alkyl groups that have ahydroxyl group appended thereto. As used herein, “hydroxyalkoxy” groupsare alkoxy groups that have a hydroxyl group appended thereto. As usedherein, “halogen” refers to fluorine, chlorine, bromine and iodine.

As used herein, the term “heteroarylalkyl” means an arylalkyl group thatcontains a heteroatom in the aryl moiety. The term “oxy” denotes thepresence of an oxygen atom. Thus, “alkoxy” groups are alkyl groups thatare attached through an oxygen atom, and “carbonyloxy” groups arecarbonyl groups that are attached through an oxygen atom.

As used herein, the terms “heterocycloalkyl” and “heterocycloalkoxy”mean an alkyl or an alkoxy group that has a heterocyclo group attachedto the alkyl moiety thereof, and the term “arylalkoxy” means an alkoxygroup that has an aryl group attached to the alkyl moiety thereof. Asused herein, the term “alkylcarbonyloxy” means a group of formula—O—C(=O)—alkyl.

As used herein, the term “alkyloxy-alkoxy” denotes an alkoxy group thatcontains an alkyloxy substituent attached to its alkyl moiety. The term“alkoxy-alkylthio” means an alkylthio group (i.e., a group of formula—S—alkyl) that contains an alkoxy substituent attached to its alkylmoiety. The term “hydroxy-alkylthio” means an alkylthio group (i.e., agroup of formula —S—alkyl) that contains a hydroxy substituent attachedto its alkyl moiety.

As used herein, the term “monosaccharide” has its accustomed meaning asa simple sugar. As used herein, the term “amino acid” denotes a moleculecontaining both an amino group and a carboxyl group. Embodiments ofamino acids include a-amino acids; i.e., carboxylic acids of generalformula HOOC—CH(NH2)-(side chain). Side chains of amino acids includenaturally occurring and non-naturally occurring moieties. Non-naturallyoccurring (i.e., unnatural) amino acid side chains are moieties that areused in place of naturally occurring amino acid side chains in, forexample, amino acid analogs. See, for example, Lehninger, Biochemistry,Second Edition, Worth Publishers, Inc, 1975, pages 73-75, incorporatedby reference herein. In certain embodiments, substituent groups for thecompounds described herein include the residue of an amino acid afterremoval of the hydroxyl moiety of the carboxyl group thereof; i.e.,groups of Formula —C(=O)CH(NH₂)— (side chain).

As used herein, “pharmaceutically acceptable salts” refer to derivativesof the disclosed compounds wherein the parent compound is modified bymaking acid or base salts thereof. Examples of pharmaceuticallyacceptable salts include, but are not limited to, mineral or organicacid salts of basic residues such as amines; alkali or organic salts ofacidic residues such as carboxylic acids; and the like. Thepharmaceutically acceptable salts include the conventional non-toxicsalts or the quaternary ammonium salts of the parent compound formed,for example, from non-toxic inorganic or organic acids. For example,such conventional non-toxic salts include those derived from inorganicacids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric,nitric and the like; and the salts prepared from organic acids such asacetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric,citric, ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic,benzoic, salicylic, sulfanilic, 2-acetoxybenzoic, fumaric,toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isethionic,and the like. The present invention is based upon the surprisingdiscovery that compounds of Formula I are able to both prevent and treatdamage to sensory hair cells. As shown herein, administration of thecompound of Formula I prevents damage to sensory hair cells. Further,the compound of Formula I has been shown to effectively treat damage tosensory hair cells or cochlear neurons.

The present invention provides a method for preventing damage to sensoryhair cells or cochlear neurons in a subject by administering aneffective amount of the compound of Formula I. The present inventionfurther provides a method for treating damage to sensory hair cells in asubject by administering a therapeutically effective amount of thecompound of Formula I.

The present invention provides a method for preventing hearing loss in asubject comprising administering to said subject an effective amount ofa fused pyrrolocarbazole of Formula I having the formula:

or a stereoisomer or pharmaceutically acceptable salt form thereof,wherein:

ring D is selected from phenyl and cyclohexene with double bond a-b;

ring B and ring F are independently selected from:

(a) a 6-membered carbocyclic ring in which from 1 to 3 carbon atoms maybe replaced by heteroatoms;

(b) a 5-membered carbocyclic ring; and

(c) a 5-membered carbocyclic ring in which either:

(1) one carbon atom is replaced with an oxygen, nitrogen, or sulfuratom;

(2) two carbon atoms are replaced with a sulfur and a nitrogen atom, anoxygen and a nitrogen atom, or two nitrogen atoms; or

(3) three carbon atoms are replaced with three nitrogen atoms, oneoxygen and two nitrogen atoms, or one sulfur and two nitrogen atoms;

G-X-W is selected from:

(a) (Z¹Z²)C—N(R¹)—C(Z¹Z²);

(b) CH(R¹)—C(=O)—N(R¹); and

(c) N(R¹)—C(=O)—CH(R¹);

Z¹ and Z², at each occurrence, are independently selected from H, H; H,OR; H, SR; H, N(R)₂; and a group wherein Z¹ and Z² together form amoiety selected from =O, =S, and =NR; with the proviso that at least oneof the pairs Z¹ and Z² forms =O;

R is independently selected from H, optionally substituted alkyl, OH,alkoxy, OC(=O)R^(1a), OC(=O)NR^(1c)R^(1d),O(CH₂)_(p)NR^(1c)R^(1d),O(CH₂)_(p)OR^(1b), optionally substituted arylalkyl and optionallysubstituted heteroarylalkyl;

R¹ is independently selected from:

(a) H, optionally substituted alkyl, optionally substituted aryl,optionally substituted arylalkyl, optionally substituted heteroaryl andoptionally substituted heteroarylalkyl;

(b) C(=O)R^(1a);

(c) OR^(1b);

(d) C(=O)NHR^(1b), NR^(1c)R^(1d), (CH₂)_(p)NR^(1c)R^(1d),(CH₂)_(p)OR^(1b), O(CH₂)_(p)OR^(1b) and O(CH₂)_(p)NR^(1c)R^(1d);

R^(1a) is independently selected from optionally substituted alkyl,optionally substituted aryl and optionally substituted heteroaryl;

R^(1b) is independently selected from H and optionally substitutedalkyl;

R^(1c) and R_(1d) are each independently selected from H, optionallysubstituted alkyl and a linking group of the formula (CH₂)₂—X¹—(CH₂)₂;

X¹ is independently selected from O, S and CH₂;

Q is selected from NR², O, S, NR²², CHR²³, X⁴CH(R²³), CH(R²³)X⁴, whereinX⁴ is selected from O, S, CH₂, NR²² and NR^(2;)

R² is selected from H, SO₂R^(2a), CO₂R^(2a), C(=O)R^(2a),C(=O)NR^(2c)R^(2d), optionally substituted alkyl, optionally substitutedalkenyl and optionally substituted alkynyl, wherein said optionalsubstituents are one to about three R⁵ groups;

R^(2a) is independently selected from optionally substituted alkyl,optionally substituted aryl, optionally substituted carbocyclyl,optionally substituted heterocyclyl, OR^(2b), CONH₂, NR^(2c)R^(2d),(CH₂)_(p)NR^(2c)R^(2d) and O(CH₂)_(p)NR^(2c)R^(2d);

R^(2b) is selected from H and optionally substituted alkyl;

R^(2c) and R^(2d) are each independently selected from H and optionallysubstituted alkyl, or together form a linking group of the formula(CH₂)₂—X¹—(CH₂)₂;

R³ and R⁴ are each independently selected from:

(a) H, aryl, heteroaryl, F, Cl, Br, I, CN, CF₃, NO₂, OH, OR⁹,O(CH₂)_(p)NR¹¹R¹², OC(=O)R⁹, OC(=O)NR¹¹R¹², O(CH₂)_(p)OR¹⁰, CH₂OR¹⁰,NR¹¹R¹², NR¹⁰S(=O)₂R⁹ and NR¹⁰C(=O)R⁹;

(b) CH₂OR¹⁴;

(c) NR¹⁰C(=O)NR¹¹R¹², CO₂R¹⁰, C(=O)R⁹, C(=O)NR¹¹R¹², CH=NOR¹⁰, CH=NR¹⁰,(CH₂)_(p)NR¹¹R¹², (CH₂)_(p)NHR¹⁴ and CH=NNR¹¹R¹²;

(d) S(O)_(y)R⁹, (CH₂)_(p)S(O)_(y)R⁹ and CH₂S(O)_(y)R¹⁴;

(e) optionally substituted alkyl, optionally substituted alkenyl andoptionally substituted alkynyl, wherein said optional substituents areone to about three R⁵ groups;

R⁹ is selected from alkyl, (CH₂)_(r)aryl and (CH₂)_(r)heteroaryl;

R¹⁰ is selected from H, alkyl, (CH₂)_(r)aryl and (CH₂)_(r)heteroaryl;

R¹¹ and R¹² are independently selected from H and optionally substitutedalkyl, or together form a linking group of the formula (CH₂)₂—X¹—(CH₂)₂;

R⁵ is selected from aryl, heteroaryl, arylalkoxy, heterocycloalkoxy,hydroxyalkoxy, alkyloxy-alkoxy, hydroxyalkylthio, alkoxy-alkylthio, F,Cl, Br, I, CN, NO₂, OH, OR⁹, X²(CH₂)_(p)NR¹¹R¹²,X²(CH₂)_(p)C(=O)NR¹¹R¹², X²(CH₂)_(p)OC(=O)NR¹¹R¹², X²(CH₂)_(p)CO₂R⁹,X²(CH₂)_(p)S(O)_(y)R⁹, X²(CH₂)_(p)NR¹⁰C(=O)NR¹¹R¹², OC(=O)R⁹,OC(=O)NHR¹⁰, O-tetrahydropyranyl, NR¹¹R¹², NR¹⁰C(=O)R⁹, NR¹⁰CO₂R⁹,NR¹⁰C(=O)NR¹¹R¹², NHC(=NH)NH₂, NR¹⁰S(O)₂R⁹, S(O)_(y)R⁹, CO₂R¹⁰,C(=O)NR¹¹R¹², C(=O)R⁹, CH₂OR¹⁰, CH=NNR¹¹R¹², CH=NOR¹⁰, CH=NR⁹,CH=NNHCH(N=NH)NH₂, S(=O)₂NR¹¹R¹², P(=O)(OR¹⁰)₂, OR¹⁴, and amonosaccharide wherein each hydroxyl group of the monosaccharide isindependently either unsubstituted or is replaced by H, alkyl,alkylcarbonyloxy, or alkoxy;

X² is O, S, or NR¹⁰;

Y is selected from:

(a) a direct bond;

(b) optionally substituted CH₂, CH₂CH₂ or CH₂CH₂CH₂, wherein saidoptional substituents are one to about three R¹⁹ groups; and

(c) CH=CH, CH(OH)—CH(OH), O, S, S(=O), S(=O)₂, C(R¹⁸)₂, C=C(R¹⁹)₂,C(=O), C(=NOR²⁰), C(OR²⁰)R²⁰, C(=O)CH(R¹⁸), CH(R¹⁸)C(=O),C(=NOR²⁰)CH(R¹⁸), CHR²¹C(=NOR²⁰), C(=O)N(R²¹), N(R²¹)C(=O), CH₂Z, ZCH₂and CH₂ZCH₂, where Z is selected from C(R²⁰)₂, O, S, CO₂R²⁰, C(=NOR²⁰)and N(R²⁰);

R¹⁸ is independently selected from H, SO₂R^(18a), CO₂R^(18a),C(=O)R^(18a), C(=O)NR^(18c)R^(18d), optionally substituted alkyl,optionally substituted alkenyl, and optionally substituted alkynyl;

R^(18a) is independently selected from optionally substituted alkyl,optionally substituted aryl, optionally substituted carbocyclyl andoptionally substituted heterocyclyl;

R^(18c) and R^(18d) are each independently selected from H andoptionally substituted alkyl, or together form a linking group of theformula (CH₂)₂—X¹—(CH₂)₂;

R¹⁹ is independently selected from R²⁰, thioalkyl, halogen, optionallysubstituted alkyl, optionally substituted alkenyl and optionallysubstituted alkynyl;

R²⁰ is independently selected from H, alkyl, OH, alkoxy, OC(=O)R^(18a),OC(=O)NR^(18c)R^(18d), OC(=S)NR^(18c)R^(18d), O(CH₂)_(p)NR^(18c)R^(18d),O(CH₂)_(p)OR²¹, optionally substituted arylalkyl, optionally substitutedheterocyclylalkyl and optionally substituted carbocyclyl;

R²¹ is independently selected from H and alkyl;

Q′ is selected from:

(a) a direct bond;

(b) NR^(6;)

(c) optionally substituted CH₂, CH₂CH₂ or CH₂CH₂CH₂;

(d) CR²²R²⁴; and

(e) CH=CH, CH(OH)CH(OH), O, S, S(=O), S(=O)₂, C(=O), C(=NOR¹¹),C(OR¹¹)(R¹²), C(=O)CH(R¹³), CH(R¹³)C(=O), C(R¹⁰)₂, C(=NOR¹¹)CH(R¹³),CH(R¹³)C(=NOR¹¹), CH₂Z′, Z′—CH₂ and CH₂Z′CH₂;

Z′ is selected from C(R¹¹)(OR¹²), O, S, C(=O), C(=NOR¹¹) and NR¹¹;

R⁶ is selected from H, SO₂R^(2a), CO₂R^(2a), C(=O)R^(2a), C(=O)NR^(1c),R^(1d), optionally substituted alkyl, optionally substituted alkenyl,and optionally substituted alkynyl, wherein said optional substituentsare one to about three R⁵ groups; or

alternatively, when Q is NR² and Q′ is NR⁶ or C(R¹⁰)₂, R² and R⁶ or oneof R¹⁰ are joined together to form:

wherein R⁷ and R⁸ are each independently selected from H, OH, alkyl,alkoxy, optionally substituted arylalkyl, optionally substitutedheteroarylalkyl, (CH₂)_(p)OR¹⁰, (CH₂)_(p)OC(=O)NR¹¹R¹² and(CH₂)_(p)NR¹¹R^(12;) or R⁷ and R⁸ together form a linking group of theformula CH₂—X³—CH₂;

X³ is a bond, O, S, or NR¹⁰;

J is selected from a bond, O, CH=CH, S, C(=O), CH(OR¹⁰), N(R¹⁰),N(OR¹⁰), CH(NR¹¹R¹²), C(=O)N(R¹⁷), N(R¹⁷)C(=O), N(S(O)_(y)R⁹),N(S(O)_(y)NR¹¹R¹²), N(C(=O)R¹⁷), C(R¹⁵R¹⁶), N⁺(O⁻)(R¹⁰), CH(OH)CH(OH)and CH(O(C=O)R⁹)CH(OC(=O)R⁹);

J′ is selected from O, S, N(R¹⁰), N⁺(O⁻)(R¹⁰), N(OR¹⁰) and CH₂;

R¹³ is selected from alkyl, aryl and arylalkyl;

R¹⁴ is the residue of an amino acid after the hydroxyl group of thecarboxyl group is removed;

R¹⁵ and R¹⁶ are independently selected from H, OH, C(=O)R¹⁰, O(C=O)R⁹,alkyl—OH, alkoxy and CO₂R¹⁰;

R¹⁷ is selected from H, alkyl, aryl and heteroaryl;

R²² is

X⁵ and X⁶ are independently selected from O, N, S, CHR²⁶, C(OH)R²⁶,C(=O) and CH₂=C;

X⁷ and X⁸ are independently selected from a bond, O, N, S, CHR²⁶,C(OH)R²⁶, C(=O) and CH₂=C;

X⁹ and X¹⁰ are independently selected from a bond, O, N, S, C(=O) andCHR²⁶;

X¹¹ is a bond or alkylene optionally substituted with NR¹¹R¹² or OR³⁰;

R²³ is selected from H, OR²⁷, SR²⁷, R²² and R²⁸;

R²⁴ is selected from R, thioalkyl, and halogen;

R²⁵ is selected from R¹ and OC(=O)NR^(1c)R^(1d);

R²⁶ is selected from H, optionally substituted alkyl and optionallysubstituted alkoxy, wherein

(1) ring G contains 0 to about 3 ring heteroatoms;

(2) any two adjacent hydroxyl groups of ring G can be joined to form adioxolane ring;

(3) any two adjacent ring carbon atoms of ring G can be joined to form afused aryl or heteroaryl ring; with the provisos that:

(a) when X¹¹ is a bond, ring G can be heteroaryl; and

(b) ring G:

(i) contains at least one carbon atom that is saturated;

(ii) does not contain two adjacent ring 0 atoms;

(iii) contains a maximum of two C(=O) groups;

R²⁷ is selected from H and alkyl;

R²⁸ is selected from aryl, arylalkyl, SO₂R²⁹, CO₂R²⁹, C(=O)R²⁹,optionally substituted alkyl, optionally substituted alkenyl andoptionally substituted alkynyl;

R²⁹ is selected from alkyl, aryl and heteroaryl;

R³⁰ is selected from H, alkyl, acyl and C(=O)NR¹¹R¹²;

m is independently selected from 0, 1, and 2;

p is independently selected from 1, 2, 3, and 4;

r is independently selected from 0, 1, and 2;

y is independently selected from 0, 1 and 2; and

z is selected from 0, 1, 2, 3 and 4;

with the provisos that at least one of Y and Q′ is a direct bond, when Yis a direct bond, Q′ is other than a direct bond, when Q′ is a directbond, Y is other than a direct bond, and when rings B and F are phenyl,G-X-W is CH₂NHC(=O), Y is a direct bond, Q is NR² and Q′ is NR⁶ where R⁶is joined with R² to form

then R³ is other than CH₂SCH₂CH₃ (in other words, the latter proviso isdirected to the compound of Formula II).

In one preferred embodiment, Y is a direct bond and Q is NR₂. In a morepreferred embodiment, ring B and ring F of the fused pyrrolocarbazoleare phenyl, G-X-W is selected from CH₂NR¹C(=O), C(=O)NR¹CH₂, andC(=O)NR¹C(=O), and Q′ is NR⁶. In an even more preferred embodiment, thefused pyrrolocarbazole has the formula:

In an even more preferred embodiment, R³ and R⁴ of the fusedpyrrolocarbazole are selected from H, alkyl, Cl, Br, CH₂OH, CH₂SOCH₂CH₃,CH₂SO₂CH₂CH₃, NHCONHC₆H₅, CH₂SCH₂CH₃, CH₂S-phenyl, CH₂S-pyridyl, CHNHCO₂CH₃, CH₂OC(=O)NHCH₂CH₃, N(CH₃)₂, CH=NNH, CH₂N(CH₃)₂, andCH₂OCH₂CH₃; R⁷ is selected from H and alkyl; and R¹⁵ and R¹⁶ areindependently selected from H, alkyl, OH, CH₂OH, alkoxy, and CO₂alkyl.In an even more preferred embodiment, the fused pyrrolocarbazole has theformula:

In another preferred embodiment, Q′ is CH₂, CH₂CH₂, S orCH(CH(CH₃)(OH)). In a more preferred embodiment, ring B and ring F ofthe fused pyrrolocarbazole are phenyl and G-X-W is selected fromCH₂NR¹C(=O), C(=O)NR¹CH₂. In an even more preferred embodiment, thefused pyrrolocarbazole has the formula

In a more preferred embodiment, R² is H, CH₂CH₂OH,CH₂CH₂NHC(=O)—C₆H₅—OH, CH₂CH₂CH₂OH, R³ and R⁴ of the fusedpyrrolocarbazole are selected from H, alkyl, Cl, Br, alkoxy, CH₂OH,CH₂SOCH₂CH₃, CH₂SO₂CH₂CH₃, NHCONHC₆H₅, CH₂SCH₂CH₃, CH₂S-phenyl,CH₂S-pyridyl, CHNHCO₂CH₃, CH₂OC(=O)NHCH₂CH₃, N(CH₃)₂, CH=NNH,CH₂N(CH₃)₂, and CH₂OCH₂CH₃; R⁷ is selected from H and alkyl; and R¹⁵ andR¹⁶ are independently selected from H, alkyl, OH, CH₂OH, alkoxy, andCO₂alkyl. In a more preffered embodiment, the fused pyrrolocarbazole hasthe formula:

In another preferred embodiment, the fused pyrrolocarbazole has theformula:

As used herein, the term “preventing”, in the context of preventinghearing loss, loss of sense of balance, death of sensory hair cells orcochlear neurons, sensorineural hearing loss, or damage to sensory haircells or cochlear neurons and the like, refers to reducing, minimizing,or completely eliminating such loss or damage. As used herein,“preventing” may include, for example, at least about a 15% reduction ofloss or damage, more preferably at least about 25%, more preferably atleast about 50%, even more preferably at least about 75%, even morepreferably at least about 80%, even more preferably at least about 85%,even more preferably at least about 90%, even more preferably at leastabout 95%, and most preferably about 100%. As used herein, the term“sensory hair cells” refers to the hair cells present in vertebrates,including the auditory sensory hair cells present in the Organ of Corti,and the vestibular sensory hair cells present in the semicircular canalsand maculae of the inner ear.

As used herein, the term “subject” refers to mammals including but notlimited to humans and primates; avians; domestic household, sport orfarm animals including dogs, cats, sheep, goats, cows, horses and pigs;lab animals including rats, mice, rabbits and guinea pigs; fish;reptiles; zoo and wild animals.

As used herein, the term “hearing loss” refers to an inability toperceive auditory stimuli that are perceivable by a normally functioningsubject.

As used herein, the term “loss of sense of balance” refers to a deficitin the vestibular system of an animal compared to the vestibular systemof a normally functioning subject.

As used herein, the term “death of sensory hair cells” refers to acessation of the ability of one or more sensory hair cells in perceivingand/or transducing sensory stimuli.

The term “administration” includes but is not limited to, oral,subbuccal, transdermal, parenteral, subcutaneous and topical. A commonrequirement for these routes of administration is efficient and easydelivery of the compound of Formula I to the target.

One mode of administration contemplated by the present invention istopical. The compound of Formula I may be administered topically in anumber of ways, including, as a cream, a lotion, an ointment, as aerosolsprays, or as drops, including but not limited to eardrops andnosedrops.

Another mode of administration of the compound of Formula I to thesubject is subbuccal through the use of tablets.

Yet another mode of administration of the compound of Formula I issubcutaneous administration.

Another mode of administration of the compound of Formula I is oral. Thecompound of Formula I may be administered orally to a subject in anumber of ways, including, but not limited to tablets, capsules andcaplets.

A preferred mode of topical administration is through the use ofeardrops. The formulation and administration of eardrops is well withinthe skill of the art. In a preferred embodiment, eardrops may becomposed of from 0.1% to 20% by weight of the compound of Formula I in asuitable carrier. Two to four drops may be administered to a patientevery four to eight hours.

A more preferred mode of administration is the direct administration ofthe compound of Formula I in situ to the Round window via a catheter.Such administration may permit the use of lower dosages (Korver et al.,21^(st) Midwinter Research Meeting of ARO, St. Petersburg Beach, Fla.,Feb. 15-19, 1998, Abstract No. 536, page 135).

The nature of the pharmaceutical composition for the administration isdependent on the mode of administration and can readily be determined byone of ordinary skill in the art. For example, for oral administration,pharmaceutical compositions may contain, in addition to the compound ofFormula I, pharmaceutically acceptable carriers, vehicles, buffers andexcipients.

As used herein, the term “effective amount,” refers to the amount of thecompounds of Formula I and Formula II required to achieve an intendedpurpose for both prophylaxis or treatment without undesirable sideeffects, such as toxicity, irritation or allergic response. Althoughindividual needs may vary, the determination of optimal ranges foreffective amounts of formulations is within the skill of the art. Humandoses can readily be extrapolated from animal studies (Katocs et al.,Chapter 27 In: Remington's Pharmaceutical Sciences, 18th Ed., Gennaro,ed., Mack Publishing Co., Easton, Pa., 1990). Generally, the dosagerequired to provide an effective amount of a formulation, which can beadjusted by one skilled in the art, will vary depending on severalfactors, including the age, health, physical condition, weight, type andextent of the disease or disorder of the recipient, frequency oftreatment, the nature of concurrent therapy, if required, and the natureand scope of the desired effect(s) (Nies et al., Chapter 3 In: Goodman &Gilman's The Pharmacological Basis of Therapeutics, 9th Ed., Hardman etal., eds., McGraw-Hill, New York, N.Y., 1996).

According to the present invention, the compound of Formula I isadministered to subjects at a dose ranging from about 0.1 μg/kg/day toabout 100 mg/kg/day, preferably at a dose from about 1 μg/kg/day toabout 25 mg/kg/day, and more preferably at a dose of about 5 mg/kg/day.Generally, lower dosages of the compound of Formula I will be initiallyadministered to a patient. Dosages may be incrementally increased untilthe desired level is achieved. It is contemplated that the compound ofFormula I can be administered topically onto the eardrum using eardrops.Also, the compound of Formula I can be administered directly to theRound window using a catheter (Leary, New York Times, Health, Sep. 1,1998).

The present invention also provides methods for preventing loss of senseof balance in a subject comprising administering to said subject aneffective amount of a fused pyrrolocarbazole of Formula I having theformula:

or a stereoisomer or pharmaceutically acceptable salt form thereof,wherein:

ring D is selected from phenyl and cyclohexene with double bond a-b;

ring B and ring F are independently selected from:

(a) a 6-membered carbocyclic ring in which from 1 to 3 carbon atoms maybe replaced by heteroatoms;

(b) a 5-membered carbocyclic ring; and

(c) a 5-membered carbocyclic ring in which either:

(1) one carbon atom is replaced with an oxygen, nitrogen, or sulfuratom;

(2) two carbon atoms are replaced with a sulfur and a nitrogen atom, anoxygen and a nitrogen atom, or two nitrogen atoms; or

(3) three carbon atoms are replaced with three nitrogen atoms, oneoxygen and two nitrogen atoms, or one sulfur and two nitrogen atoms;

G-X-W is selected from:

(a) (Z¹Z²)C—N(R¹)—C(Z¹Z²);

(b) CH(R¹)—C(═O)—N(R¹); and

(c) N(R¹)—C(═O)—CH(R¹);

Z¹ and Z², at each occurrence, are independently selected from H, H; H,OR; H, SR; H, N(R)₂; and a group wherein Z¹ and Z² together form amoiety selected from ═O, ═S, and ═NR; with the proviso that at least oneof the pairs Z¹ and Z² forms ═O;

R is independently selected from H, optionally substituted alkyl, OH,alkoxy, OC(═O)R^(1a), OC(═O)NR^(1c)R^(1d), O(CH₂)_(p)NR^(1c)R^(1d),O(CH₂)_(p)OR^(1b), optionally substituted arylalkyl and optionallysubstituted heteroarylalkyl;

R¹ is independently selected from:

(a) H, optionally substituted alkyl, optionally substituted aryl,optionally substituted arylalkyl, optionally substituted heteroaryl andoptionally substituted heteroarylalkyl;

(b) C(═O)R^(1a);

(c) OR^(1b);

(d) C(═O)NHR^(1b), NR^(1c)R^(1d), (CH₂)_(p)NR^(1c)R^(1d),(CH₂)_(p)OR^(1b), O(CH₂)_(p)OR^(1b) and O(CH₂)_(p)NR^(1c)R^(1d);

R^(1a) is independently selected from optionally substituted alkyl,optionally substituted aryl and optionally substituted heteroaryl;

R^(1b) is independently selected from H and optionally substitutedalkyl;

R^(1c) and R^(1d) are each independently selected from H, optionallysubstituted alkyl and a linking group of the formula (CH₂)₂—X¹—(CH₂)₂;

X¹ is independently selected from O, S and CH₂;

Q is selected from NR², O, S, NR²², CHR²³, X⁴CH(R²³), CH(R²³)X⁴, whereinX⁴ is selected from O, S, CH₂, NR²² and NR²;

R² is selected from H, SO₂R^(2a), CO₂R^(2a), C(═O)R^(2a),C(═O)NR^(2c)R^(2d), optionally substituted alkyl, optionally substitutedalkenyl and optionally substituted alkynyl, wherein said optionalsubstituents are one to about three R⁵ groups;

R^(2a) is independently selected from optionally substituted alkyl,optionally substituted aryl, optionally substituted carbocyclyl,optionally substituted heterocyclyl, OR^(2b), CONH₂, NR^(2c)R^(2d),(CH₂)_(p)NR^(2c)R^(2d) and O(CH₂)_(p)NR^(2c)R^(2d);

R^(2b) is selected from H and optionally substituted alkyl;

R^(2c) and R^(2d) are each independently selected from H and optionallysubstituted alkyl, or together form a linking group of the formula(CH₂)₂—X¹—(CH₂)₂;

R³ and R⁴ are each independently selected from:

(a) H, aryl, heteroaryl, F, Cl, Br, I, CN, CF₃, NO₂, OH, OR⁹,O(CH₂)_(p)NR¹¹R¹², OC(═O)R⁹, OC(═O)NR¹¹R¹², O(CH₂)_(p)OR¹⁰, CH₂OR¹⁰,NR¹¹R¹², NR¹⁰S(═O)₂R⁹ and NR¹⁰C(═O)R⁹;

(b) CH₂OR¹⁴;

(c) NR¹⁰C(═O)NR¹¹R¹², CO₂R¹⁰, C(═O)R⁹, C(═O)NR¹¹R¹², CH═NOR¹⁰, CH═NR¹⁰,(CH₂)_(p)NR¹¹R¹², (CH₂)_(p)NHR¹⁴ and CH═NNR¹¹R¹²;

(d) S(O)_(y)R⁹, (CH₂)_(p)S(O)_(y)R⁹ and CH₂S(O)_(y)R¹⁴;

(e) optionally substituted alkyl, optionally substituted alkenyl andoptionally substituted alkynyl, wherein said optional substituents areone to about three R⁵ groups;

R⁹ is selected from alkyl, (CH₂)_(r)aryl and (CH₂)_(r)heteroaryl;

R¹⁰ is selected from H, alkyl, (CH₂)_(r)aryl and (CH₂)_(r)heteroaryl;

R¹¹ and R¹² are independently selected from H and optionally substitutedalkyl, or together form a linking group of the formula (CH₂)₂—X¹—(CH₂)₂;

R⁵ is selected from aryl, heteroaryl, arylalkoxy, heterocycloalkoxy,hydroxyalkoxy, alkyloxy-alkoxy, hydroxyalkylthio, alkoxy-alkylthio, F,Cl, Br, I, CN, NO₂, OH, OR⁹, X²(CH₂)_(p)NR¹¹R¹²,X²(CH₂)_(p)C(═O)NR¹¹R¹², X²(CH₂)_(p)OC(═O)NR¹¹R¹², X²(CH₂)_(p)CO₂R⁹,X²(CH₂)_(p)S(O)_(y)R⁹, X²(CH₂)_(p)NR¹⁰C(═O)NR¹¹R¹², OC(═O)R⁹,OC(═O)NHR¹⁰, O-tetrahydropyranyl, NR¹¹R¹², NR¹⁰C(═O)R⁹, NR¹⁰CO₂R⁹,NR¹⁰C(═O)NR¹¹R¹², NHC(═NH)NH₂, NR¹⁰S(O)₂R⁹, S(O)_(y)R⁹, CO₂R¹⁰,C(═O)NR¹¹R¹², C(═O)R⁹, CH₂OR¹⁰, CH═NNR¹¹R¹², CH═NOR¹⁰, CH═NR⁹,CH═NNHCH(N═NH)NH₂, S(═O)₂NR¹¹R¹², P(═O)(OR¹⁰)₂, OR¹⁴, and amonosaccharide wherein each hydroxyl group of the monosaccharide isindependently either unsubstituted or is replaced by H, alkyl,alkylcarbonyloxy, or alkoxy;

X² is O, S, or NR¹⁰;

Y is selected from:

(a) a direct bond;

(b) optionally substituted CH₂, CH₂CH₂ or CH₂CH₂CH₂, wherein saidoptional substituents are one to about three R¹⁹ groups; and

(c) CH═CH, CH(OH)—CH(OH), O, S, S(═O), S(═O)₂, C(R¹⁸)₂, C═C(R¹⁹)₂,C(═O), C(═NOR²⁰), C(OR²⁰)R²⁰, C(═O)CH(R¹⁸), CH(R¹⁸)C(═O),C(═NOR²⁰)CH(R¹⁸), CHR²¹C(═NOR²⁰), C(═O)N(R²¹), N(R²¹)C(═O), CH₂Z, ZCH₂and CH₂ZCH₂, where Z is selected from C(R²⁰)₂, O, S, CO₂R²⁰, C(═NOR²⁰)and N(R²⁰);

R¹⁸ is independently selected from H, SO₂R^(18a), CO₂R¹⁸, C(═O)R^(18a),C(═O)NR^(18c)R^(18d), optionally substituted alkyl, optionallysubstituted alkenyl, and optionally substituted alkynyl;

R^(18a) is independently selected from optionally substituted alkyl,optionally substituted aryl, optionally substituted carbocyclyl andoptionally substituted heterocyclyl;

R^(18c) and R^(18d) are each independently selected from H andoptionally substituted alkyl, or together form a linking group of theformula (CH₂)₂—X¹—(CH₂)₂;

R¹⁹ is independently selected from R²⁰, thioalkyl, halogen, optionallysubstituted alkyl, optionally substituted alkenyl and optionallysubstituted alkynyl;

R²⁰ is independently selected from H, alkyl, OH, alkoxy, OC(═O)R^(18a),OC(═O)NR^(18c)R^(18d), OC(═S)NR^(18c)R^(18d), O(CH₂)_(p)NR^(18c)R^(18d),O(CH₂)_(p)OR²¹, optionally substituted arylalkyl, optionally substitutedheterocyclylalkyl and optionally substituted carbocyclyl;

R²¹ is independently selected from H and alkyl;

Q′ is selected from:

(a) a direct bond;

(b) NR⁶;

(c) optionally substituted CH₂, CH₂CH₂ or CH₂CH₂CH₂;

(d) CR²²R²⁴; and

(e) CH═CH, CH(OH)CH(OH), O, S, S(═O), S(═O)₂, C(═O), C(═NOR¹¹),C(OR¹¹)(R¹²), C(═O)CH(R¹³), CH(R¹³)C(═O), C(R¹⁰)₂, C(═NOR¹¹)CH(R¹³),CH(R¹³)C(═NOR¹¹), CH₂Z′, Z′—CH₂ and CH₂Z′CH₂;

Z′ is selected from C(R¹¹)(OR¹²), O, S, C(═O), C(═NOR¹¹) and NR¹¹;

R⁶ is selected from H, SO₂R^(2a), CO₂R^(2a), C(═O)R^(2a),C(═O)NR^(1c)R^(1d), optionally substituted alkyl, optionally substitutedalkenyl, and optionally substituted alkynyl, wherein said optionalsubstituents are one to about three R⁵ groups; or

alternatively, when Q is NR² and Q′ is NR⁶ or C(R¹⁰)₂, R² and R⁶ or oneof R¹⁰ are joined together to form:

wherein R⁷ and R⁸ are each independently selected from H, OH, alkyl,alkoxy, optionally substituted arylalkyl, optionally substitutedheteroarylalkyl, (CH₂)_(p)OR¹⁰, (CH₂)_(p)OC(═O)NR¹¹R¹² and(CH₂)_(p)NR¹¹R¹²; or R⁷ and R⁸ together form a linking group of theformula CH₂—X³—CH₂;

X³ is a bond, O, S, or NR¹⁰;

J is selected from a bond, O, CH═CH, S, C(═O), CH(OR¹⁰), N(R¹⁰),N(OR¹⁰), CH(NR¹¹R¹²), C(═O)N(R¹⁷), N(R¹⁷)C(═O), N(S(O)_(y)R⁹),N(S(O)_(y)NR¹¹R¹²), N(C(═O)R¹⁷), C(R¹⁵R¹⁶), N⁺(O⁻)(R¹⁰), CH(OH)CH(OH)and CH(O(C═O)R⁹)CH(OC(═O)R⁹);

J′ is selected from O, S, N(R¹⁰), N⁺(O⁻)(R¹⁰), N(OR¹⁰) and CH₂;

R¹³ is selected from alkyl, aryl and arylalkyl;

R¹⁴ is the residue of an amino acid after the hydroxyl group of thecarboxyl group is removed;

R¹⁵ and R¹⁶ are independently selected from H, OH, C(═O)R¹⁰, O(C═O)R⁹,alkyl-OH, alkoxy and CO₂R¹⁰;

R¹⁷ is selected from H, alkyl, aryl and heteroaryl;

R²² is

X⁵ and X⁶ are independently selected from O, N, S, CHR²⁶, C(OH)R²⁶,C(═O) and CH₂═C;

X⁷ and X⁸ are independently selected from a bond, O, N, S, CHR²⁶,C(OH)R²⁶, C(═O) and CH₂═C;

X⁹ and X¹⁰ are independently selected from a bond, O, N, S, C(═O) andCHR²⁶;

X¹¹ is a bond or alkylene optionally substituted with NR¹¹R¹² or OR³⁰;

R²³ is selected from H, OR²⁷, SR²⁷, R²² and R²⁸;

R²⁴ is selected from R, thioalkyl, and halogen;

R²⁵ is selected from R¹ and OC(═O)NR^(1c)R^(1d);

R²⁶ is selected from H, optionally substituted alkyl and optionallysubstituted alkoxy, wherein

(1) ring G contains 0 to about 3 ring heteroatoms;

(2) any two adjacent hydroxyl groups of ring G can be joined to form adioxolane ring;

(3) any two adjacent ring carbon atoms of ring G can be joined to form afused aryl or heteroaryl ring; with the provisos that:

(a) when X¹¹ is a bond, ring G can be heteroaryl; and

(b) ring G:

(i) contains at least one carbon atom that is saturated;

(ii) does not contain two adjacent ring O atoms;

(iii) contains a maximum of two C(═O) groups;

R²⁷ is selected from H and alkyl;

R²⁸ is selected from aryl, arylalkyl, SO₂R²⁹, CO₂R²⁹, C(═O)R²⁹,optionally substituted alkyl, optionally substituted alkenyl andoptionally substituted alkynyl;

R²⁹ is selected from alkyl, aryl and heteroaryl;

R³⁰ is selected from H, alkyl, acyl and C(═O)NR¹¹R¹²;

m is independently selected from 0, 1, and 2;

p is independently selected from 1, 2, 3, and 4;

r is independently selected from 0, 1, and 2;

y is independently selected from 0, 1 and 2; and

z is selected from 0, 1, 2, 3 and 4;

with the provisos that at least one of Y and Q′ is a direct bond, when Yis a direct bond, Q′ is other than a direct bond, when Q′ is a directbond, Y is other than a direct bond, and when rings B and F are phenyl,G-X-W is CH₂NHC(═O), Y is a direct bond, Q is NR² and Q′ is NR⁶ where R⁶is joined with R² to form

then R³ is other than CH₂SCH₂CH₃.

The present invention also provides methods for preventing death ofsensory hair cells in a subject, said method comprising administering tosaid subject an effective amount of a fused pyrrolocarbazole of FormulaI having the formula:

or a stereoisomer or pharmaceutically acceptable salt form thereof,wherein:

ring D is selected from phenyl and cyclohexene with double bond a-b;

ring B and ring F are independently selected from:

(a) a 6-membered carbocyclic ring in which from 1 to 3 carbon atoms maybe replaced by heteroatoms;

(b) a 5-membered carbocyclic ring; and

(c) a 5-membered carbocyclic ring in which either:

(1) one carbon atom is replaced with an oxygen, nitrogen, or sulfuratom;

(2) two carbon atoms are replaced with a sulfur and a nitrogen atom, anoxygen and a nitrogen atom, or two nitrogen atoms; or

(3) three carbon atoms are replaced with three nitrogen atoms, oneoxygen and two nitrogen atoms, or one sulfur and two nitrogen atoms;

G-X-W is selected from:

(a) (Z¹Z²)C—N(R¹)—C(Z¹Z²);

(b) CH(R¹)—C(═O)—N(R¹); and

(c) N(R¹)—C(═O)—CH(R¹);

Z¹ and Z², at each occurrence, are independently selected from H, H; H,OR; H, SR; H, N(R)₂; and a group wherein Z¹ and Z² together form amoiety selected from ═O, ═S, and ═NR; with the proviso that at least oneof the pairs Z¹ and Z² forms ═O;

R is independently selected from H, optionally substituted alkyl, OH,alkoxy, OC(═O)R^(1a), OC(═O)NR^(1c)R^(1d), O(CH₂)_(p)NR^(1c)R^(1d),O(CH₂)_(p)OR^(1b), optionally substituted arylalkyl and optionallysubstituted heteroarylalkyl;

R¹ is independently selected from:

(a) H, optionally substituted alkyl, optionally substituted aryl,optionally substituted arylalkyl, optionally substituted heteroaryl andoptionally substituted heteroarylalkyl;

(b) C(═O)R^(1a);

(c) OR^(1b);

(d) C(═O)NHR^(1b), NR^(1c)R^(1d), (CH₂)_(p)NR^(1c)R^(1d),(CH₂)_(p)OR^(1b), O(CH₂)_(p)OR^(1b) and O(CH₂)_(p)NR^(1c)R^(1d);

R^(1a) is independently selected from optionally substituted alkyl,optionally substituted aryl and optionally substituted heteroaryl;

R^(1b) is independently selected from H and optionally substitutedalkyl;

R^(1c) and R^(1d) are each independently selected from H, optionallysubstituted alkyl and a linking group of the formula (CH₂)₂—X¹—(CH₂)₂;

X¹ is independently selected from O, S and CH₂;

Q is selected from NR², O, S, NR²², CHR²³, X⁴CH(R²³), CH(R²³)X⁴, whereinX⁴ is selected from O, S, CH₂, NR²² and NR²;

R² is selected from H, SO₂R^(2a), CO₂R^(2a), C(═O)R^(2a),C(═O)NR^(2c)R^(2d), optionally substituted alkyl, optionally substitutedalkenyl and optionally substituted alkynyl, wherein said optionalsubstituents are one to about three R⁵ groups;

R^(2a) is independently selected from optionally substituted alkyl,optionally substituted aryl, optionally substituted carbocyclyl,optionally substituted heterocyclyl, OR^(2b), CONH₂, NR^(2c)R^(2d),(CH₂)_(p)NR^(2c)R^(2d) and O(CH₂)_(p)NR^(2c)R^(2d);

R^(2b) is selected from H and optionally substituted alkyl;

R^(2c) and R^(2d) are each independently selected from H and optionallysubstituted alkyl, or together form a linking group of the formula(CH₂)₂—X¹—(CH₂)₂;

R³ and R⁴ are each independently selected from:

(a) H, aryl, heteroaryl, F, Cl, Br, I, CN, CF₃, NO₂, OH, OR⁹,O(CH₂)_(p)NR¹¹R¹², OC(═O)R⁹, OC(═O)NR¹¹R¹², O(CH₂)_(p)OR¹⁰, CH₂OR¹⁰,NR¹¹R¹², NR¹⁰S(═O)₂R⁹ and NR¹⁰C(═O)R⁹;

(b) CH₂OR¹⁴;

(c) NR¹⁰C(═O)NR¹¹R¹², CO₂R¹⁰, C(═O)R⁹, C(═O)NR¹¹R¹², CH═NOR¹⁰, CH═NR¹⁰,(CH₂)_(p)NR¹¹R¹², (CH₂)_(p)NHR¹⁴ and CH═NNR¹¹R¹²;

(d) S(O)_(y)R⁹, (CH₂)_(p)S(O)_(y)R⁹ and CH₂S(O)_(y)R¹⁴;

(e) optionally substituted alkyl, optionally substituted alkenyl andoptionally substituted alkynyl, wherein said optional substituents areone to about three R⁵ groups;

R⁹ is selected from alkyl, (CH₂)_(r)aryl and (CH₂)_(r)heteroaryl;

R¹⁰ is selected from H, alkyl, (CH₂)_(r)aryl and (CH₂)_(r)heteroaryl;

R¹¹ and R¹² are independently selected from H and optionally substitutedalkyl, or together form a linking group of the formula (CH₂)₂—X¹—(CH₂)₂;

R⁵ is selected from aryl, heteroaryl, arylalkoxy, heterocycloalkoxy,hydroxyalkoxy, alkyloxy-alkoxy, hydroxyalkylthio, alkoxy-alkylthio, F,Cl, Br, I, CN, NO₂, OH, OR⁹, X²(CH₂)_(p)NR¹¹R¹²,X²(CH₂)_(p)C(═O)NR¹¹R¹², X²(CH₂)_(p)OC(═O)NR¹¹R¹², X²(CH₂)_(p)CO₂R⁹,X²(CH₂)_(p)S(O)_(y)R⁹, X²(CH₂)_(p)NR¹⁰C(═O)NR¹¹R¹², OC(═O)R⁹,OC(═O)NHR¹⁰, O-tetrahydropyranyl, NR¹¹R¹², NR¹⁰C(═O)R⁹, NR¹⁰CO₂R⁹,NR¹⁰C(═O)NR¹¹R¹², NHC(═NH)NH₂, NR¹⁰S(O)₂R⁹, S(O)_(y)R⁹, CO₂R¹⁰,C(═O)NR¹¹R¹², C(═O)R⁹, CH₂OR¹⁰, CH═NNR¹¹R¹², CH═NOR¹⁰, CH═NR⁹,CH═NNHCH(N═NH)NH₂, S(═O)₂NR¹¹R¹², P(═O)(OR¹⁰)₂, OR¹⁴, and amonosaccharide wherein each hydroxyl group of the monosaccharide isindependently either unsubstituted or is replaced by H, alkyl,alkylcarbonyloxy, or alkoxy;

X² is O, S, or NR¹⁰;

Y is selected from:

(a) a direct bond;

(b) optionally substituted CH₂, CH₂CH₂ or CH₂CH₂CH₂, wherein saidoptional substituents are one to about three R¹⁹ groups; and

(c) CH═CH, CH(OH)—CH(OH), O, S, S(═O), S(═O)₂, C(R¹⁸)₂, C═C(R¹⁹)₂,C(═O), C(═NOR²⁰), C(OR²⁰)R²⁰, C(═O)CH(R¹⁸), CH(R¹⁸)C(═O),C(═NOR²⁰)CH(R¹⁸), CHR²¹C(═NOR²⁰) C(═O)N(R²¹), N(R²¹)C(═O), CH₂Z, ZCH₂and CH₂ZCH₂, where Z is selected from C(R²⁰)₂, O, S, CO₂R²⁰, C(═NOR²⁰)and N(R²⁰);

R¹⁸ is independently selected from H, SO₂R^(18a), CO₂R^(18a),C(═O)R^(18a), C(═O)NR^(18c)R^(18d), optionally substituted alkyl,optionally substituted alkenyl, and optionally substituted alkynyl;

R^(18a) is independently selected from optionally substituted alkyl,optionally substituted aryl, optionally substituted carbocyclyl andoptionally substituted heterocyclyl;

R^(18c) and R^(18d) are each independently selected from H andoptionally substituted alkyl, or together form a linking group of theformula (CH₂)₂—X¹—(CH₂)₂;

R¹⁹ is independently selected from R²⁰, thioalkyl, halogen, optionallysubstituted alkyl, optionally substituted alkenyl and optionallysubstituted alkynyl;

R²⁰ is independently selected from H, alkyl, OH, alkoxy, OC(═O)R^(18a),OC(═O)NR^(18c)R^(18d), OC(═S)NR^(18c)R^(18d), O(CH₂)_(p)NR^(18c)R^(18d),O(CH₂)_(p)OR²¹, optionally substituted arylalkyl, optionally substitutedheterocyclylalkyl and optionally substituted carbocyclyl;

R²¹ is independently selected from H and alkyl;

Q′ is selected from:

(a) a direct bond;

(b) NR⁶;

(c) optionally substituted CH₂, CH₂CH₂ or CH₂CH₂CH₂;

(d) CR²²R²⁴; and

(e) CH═CH, CH(OH)CH(OH), O, S, S(═O), S(═O)₂, C(═O), C(═NOR¹¹),C(OR¹¹)(R¹²), C(═O)CH(R¹³), CH(R¹³)C(═O), C(R¹⁰)₂, C(═NOR¹¹)CH(R¹³),CH(R¹³)C(═NOR¹¹), CH₂Z′, Z′—CH₂ and CH₂Z′CH₂;

Z′ is selected from C(R¹¹)(OR¹²), O, S, C(═O), C(═NOR¹¹) and NR¹¹;

R⁶ is selected from H, SO₂R^(2a), CO₂R^(2a), C(═O)R^(2a),C(═O)NR^(1c)R^(1d), optionally substituted alkyl, optionally substitutedalkenyl, and optionally substituted alkynyl, wherein said optionalsubstituents are one to about three R⁵ groups; or

alternatively, when Q is NR² and Q′ is NR⁶ or C(R¹⁰)₂, R² and R⁶ or oneof R¹⁰ are joined together to form:

wherein R⁷ and R⁸ are each independently selected from H, OH, alkyl,alkoxy, optionally substituted arylalkyl, optionally substitutedheteroarylalkyl, (CH₂)_(p)OR¹⁰, (CH₂)_(p)OC(═O)NR¹¹R¹² and(CH₂)_(p)NR¹¹R¹²; or R⁷ and R⁸ together form a linking group of theformula CH₂—X³—CH₂;

X³ is a bond, O, S, or NR¹⁰;

J is selected from a bond, O, CH═CH, S, C(═O), CH(OR¹⁰), N(R¹⁰),N(OR¹⁰), CH(NR¹¹R¹²), C(═O)N(R¹⁷), N(R¹⁷)C(═O), N(S(O)_(y)R⁹),N(S(O)_(y)NR¹¹R¹²), N(C(═O)R¹⁷), C(R¹⁵R¹⁶), N⁺(O⁻)(R¹⁰), CH(OH)CH(OH)and CH(O(C═O)R⁹)CH(OC(═O)R⁹);

J′ is selected from O, S, N(R¹⁰), N⁺(O⁻)(R¹⁰), N(OR¹⁰) and CH₂;

R¹³ is selected from alkyl, aryl and arylalkyl;

R¹⁴ is the residue of an amino acid after the hydroxyl group of thecarboxyl group is removed;

R¹⁵ and R¹⁶ are independently selected from H, OH, C(═O)R¹⁰, O(C═O)R⁹,alkyl-OH, alkoxy and CO₂R¹⁰;

R¹⁷ is selected from H, alkyl, aryl and heteroaryl;

R²² is

X⁵ and X⁶ are independently selected from O, N, S, CHR²⁶, C(OH)R²⁶,C(═O) and CH₂═C;

X⁷ and X⁸ are independently selected from a bond, O, N, S, CHR²⁶,C(OH)R²⁶, C(═O) and CH₂═C;

X⁹ and X¹⁰ are independently selected from a bond, O, N, S, C(═O) andCHR²⁶;

X¹¹ is a bond or alkylene optionally substituted with NR¹¹R¹² or OR³⁰;

R²³ is selected from H, OR²⁷, SR²⁷, R²² and R²⁸;

R²⁴ is selected from R, thioalkyl, and halogen;

R²⁵ is selected from R¹ and OC(═O)NR^(1c)R^(1d);

R²⁶ is selected from H, optionally substituted alkyl and optionallysubstituted alkoxy, wherein

(1) ring G contains 0 to about 3 ring heteroatoms;

(2) any two adjacent hydroxyl groups of ring G can be joined to form adioxolane ring;

(3) any two adjacent ring carbon atoms of ring G can be joined to form afused aryl or heteroaryl ring; with the provisos that:

(a) when X¹¹ is a bond, ring G can be heteroaryl; and

(b) ring G:

(i) contains at least one carbon atom that is saturated;

(ii) does not contain two adjacent ring O atoms;

(iii) contains a maximum of two C(═O) groups;

R²⁷ is selected from H and alkyl;

R²⁸ is selected from aryl, arylalkyl, SO₂R²⁹, CO₂R²⁹, C(═O)R²⁹,optionally substituted alkyl, optionally substituted alkenyl andoptionally substituted alkynyl;

R²⁹ is selected from alkyl, aryl and heteroaryl;

R³⁰ is selected from H, alkyl, acyl and C(═O)NR¹¹R¹²;

m is independently selected from 0, 1, and 2;

p is independently selected from 1, 2, 3, and 4;

r is independently selected from 0, 1, and 2;

y is independently selected from 0, 1 and 2; and

z is selected from 0, 1, 2, 3 and 4;

with the provisos that at least one of Y and Q′ is a direct bond, when Yis a direct bond, Q′ is other than a direct bond, when Q′ is a directbond, Y is other than a direct bond, and when rings B and F are phenyl,G-X-W is CH₂NHC(═O), Y is a direct bond, Q is NR² and Q′ is NR⁶ where R⁶is joined with R² to form

then R³ is other than CH₂SCH₂CH₃.

The present invention also provides methods for preventing suddensensorineural hearing loss in a subject due to death of sensory haircells comprising administering to said subject an effective amount of afused pyrrolocarbazole of Formula I having the formula:

or a stereoisomer or pharmaceutically acceptable salt form thereof,wherein:

ring D is selected from phenyl and cyclohexene with double bond a-b;

ring B and ring F are independently selected from:

(a) a 6-membered carbocyclic ring in which from 1 to 3 carbon atoms maybe replaced by heteroatoms;

(b) a 5-membered carbocyclic ring; and

(c) a 5-membered carbocyclic ring in which either:

(1) one carbon atom is replaced with an oxygen, nitrogen, or sulfuratom;

(2) two carbon atoms are replaced with a sulfur and a nitrogen atom, anoxygen and a nitrogen atom, or two nitrogen atoms; or

(3) three carbon atoms are replaced with three nitrogen atoms, oneoxygen and two nitrogen atoms, or one sulfur and two nitrogen atoms;

G-X-W is selected from:

(a) (Z¹Z²)C—N(R¹)—C(Z¹Z²);

(b) CH(R¹)—C(═O)—N(R¹); and

(c) N(R¹)—C(═O)—CH(R¹);

Z¹ and Z², at each occurrence, are independently selected from H, H; H,OR; H, SR; H, N(R)₂; and a group wherein Z¹ and Z² together form amoiety selected from ═O, ═S, and ═NR; with the proviso that at least oneof the pairs Z¹ and Z² forms ═O;

R is independently selected from H, optionally substituted alkyl, OH,alkoxy, OC(═O)R^(1a), OC(═O)NR^(1c)R^(1d), O(CH₂)_(p)NR^(1c)R^(1d),O(CH₂)_(p)OR^(1b), optionally substituted arylalkyl and optionallysubstituted heteroarylalkyl;

R¹ is independently selected from:

(a) H, optionally substituted alkyl, optionally substituted aryl,optionally substituted arylalkyl, optionally substituted heteroaryl andoptionally substituted heteroarylalkyl;

(b) C(═O)R^(1a);

(c) OR^(1b);

(d) C(═O)NHR^(1b), NR^(1c)R^(1d), (CH₂)_(p)NR^(1c)R^(1d),(CH₂)_(p)OR^(1b), O(CH₂)_(p)OR^(1b) and O(CH₂)_(p)NR^(1c)R^(1d);

R^(1a) is independently selected from optionally substituted alkyl,optionally substituted aryl and optionally substituted heteroaryl;

R^(1b) is independently selected from H and optionally substitutedalkyl;

R^(1c) and R^(1d) are each independently selected from H, optionallysubstituted alkyl and a linking group of the formula (CH₂)₂—X¹—(CH₂)₂;

X¹ is independently selected from O, S and CH₂;

Q is selected from NR², O, S, NR²², CHR²³, X⁴CH(R²³), CH(R²³)X⁴, whereinX⁴ is selected from O, S, CH₂, NR²² and NR²;

R² is selected from H, SO₂R^(2a), CO₂R^(2a), C(═O)R^(2a),C(═O)NR^(2c)R^(2d), optionally substituted alkyl, optionally substitutedalkenyl and optionally substituted alkynyl, wherein said optionalsubstituents are one to about three R⁵ groups;

R^(2a) is independently selected from optionally substituted alkyl,optionally substituted aryl, optionally substituted carbocyclyl,optionally substituted heterocyclyl, OR^(2b), CONH₂, NR^(2c)R^(2d),(CH₂)_(p)NR^(2c)R^(2d) and O(CH₂)_(p)NR^(2c)R^(2d);

R^(2b) is selected from H and optionally substituted alkyl;

R^(2c) and R^(2d) are each independently selected from H and optionallysubstituted alkyl, or together form a linking group of the formula(CH₂)₂—X¹—(CH₂)₂;

R³ and R⁴ are each independently selected from:

(a) H, aryl, heteroaryl, F, Cl, Br, I, CN, CF₃, NO₂, OH, OR⁹,O(CH₂)_(p)NR¹¹R¹², OC(═O)R⁹, OC(═O)NR¹¹R¹², O(CH₂)_(p)OR¹⁰, CH₂OR¹⁰,NR¹¹R¹², NR¹⁰S(═O)₂R⁹ and NR¹⁰C(═O)R⁹;

(b) CH₂OR¹⁴;

(c) NR¹⁰C(═O)NR¹¹R¹², CO₂R¹⁰, C(═O)R⁹, C(═O)NR¹¹R¹², CH═NOR¹⁰, CH═NR¹⁰,(CH₂)_(p)NR¹¹R¹², (CH₂)_(p)NHR¹⁴ and CH═NNR¹¹R¹²;

(d) S(O)_(y)R⁹, (CH₂)_(p)S(O)_(y)R⁹ and CH₂S(O)_(y)R¹⁴;

(e) optionally substituted alkyl, optionally substituted alkenyl andoptionally substituted alkynyl, wherein said optional substituents areone to about three R⁵ groups;

R⁹ is selected from alkyl, (CH₂)_(r)aryl and (CH₂)_(r)heteroaryl;

R¹⁰ is selected from H, alkyl, (CH₂)_(r)aryl and (CH₂)_(r)heteroaryl;

R¹¹ and R¹² are independently selected from H and optionally substitutedalkyl, or together form a linking group of the formula (CH₂)₂—X¹—(CH₂)₂;

R⁵ is selected from aryl, heteroaryl, arylalkoxy, heterocycloalkoxy,hydroxyalkoxy, alkyloxy-alkoxy, hydroxyalkylthio, alkoxy-alkylthio, F,Cl, Br, I, CN, NO₂, OH, OR⁹, X²(CH₂)_(p)NR¹¹R¹²,X²(CH₂)_(p)C(═O)NR¹¹R¹², X²(CH₂)_(p)OC(═O)NR¹¹R¹², X²(CH₂)_(p)CO₂R⁹,X²(CH₂)_(p)S(O)_(y)R⁹, X²(CH₂)_(p)NR¹⁰C(═O)NR¹¹R¹², OC(═O)R⁹,OC(═O)NHR¹⁰, O-tetrahydropyranyl, NR¹¹R¹², NR¹⁰C(═O)R⁹, NR¹⁰CO₂R⁹,NR¹⁰C(═O)NR¹¹R¹², NHC(═NH)NH₂, NR¹⁰S(O)₂R⁹, S(O)_(y)R⁹, CO₂R¹⁰,C(═O)NR¹¹R¹², C(═O)R⁹, CH₂OR¹⁰, CH═NNR¹¹R¹², CH═NOR¹⁰, CH═NR⁹,CH═NNHCH(N═NH)NH₂, S(═O)₂NR¹¹R¹², P(═O)(OR¹⁰)₂, OR¹⁴, and amonosaccharide wherein each hydroxyl group of the monosaccharide isindependently either unsubstituted or is replaced by H, alkyl,alkylcarbonyloxy, or alkoxy;

X² is O, S, or NR¹⁰;

Y is selected from:

(a) a direct bond;

(b) optionally substituted CH₂, CH₂CH₂ or CH₂CH₂CH₂, wherein saidoptional substituents are one to about three R¹⁹ groups; and

(c) CH═CH, CH(OH)—CH(OH), O, S, S(═O), S(═O)₂, C(R¹⁸)₂, C═C(R¹⁹)₂,C(═O), C(═NOR²⁰), C(OR²⁰)R²⁰, C(═O)CH(R¹⁸), CH(R¹⁸)C(═O),C(═NOR²⁰)CH(R¹⁸), CHR²¹C(═NOR²⁰), C(═O)N(R²¹), N(R²¹)C(═O), CH₂Z, ZCH₂and CH₂ZCH₂, where Z is selected from C(R²⁰)₂, O, S, CO₂R²⁰, C(═NOR²⁰)and N(R²⁰);

R¹⁸ is independently selected from H, SO₂R^(18a), CO₂R^(18a),C(═O)R^(18a), C(═O)NR^(18c)R^(18d), optionally substituted alkyl,optionally substituted alkenyl, and optionally substituted alkynyl;

R^(18a) is independently selected from optionally substituted alkyl,optionally substituted aryl, optionally substituted carbocyclyl andoptionally substituted heterocyclyl;

R^(18c) and R^(18d) are each independently selected from H andoptionally substituted alkyl, or together form a linking group of theformula (CH₂)₂—X¹—(CH₂)₂;

R¹⁹ is independently selected from R²⁰, thioalkyl, halogen, optionallysubstituted alkyl, optionally substituted alkenyl and optionallysubstituted alkynyl;

R²⁰ is independently selected from H, alkyl, OH, alkoxy, OC(═O)R^(18a),OC(═O)NR^(18c)R^(18d), OC(═S)NR^(18c)R^(18d), O(CH₂)_(p)NR^(18c)R^(18d),O(CH₂)_(p)OR²¹, optionally substituted arylalkyl, optionally substitutedheterocyclylalkyl and optionally substituted carbocyclyl;

R²¹ is independently selected from H and alkyl;

Q′ is selected from:

(a) a direct bond;

(b) NR⁶;

(c) optionally substituted CH₂, CH₂CH₂ or CH₂CH₂CH₂;

(d) CR²²R²⁴; and

(e) CH═CH, CH(OH)CH(OH), O, S, S(═O), S(═O)₂, C(═O), C(═NOR¹¹),C(OR¹¹)(R¹²), C(═O)CH(R¹³), CH(R¹³)C(═O), C(R¹⁰)₂, C(═NOR¹¹)CH(R¹³),CH(R¹³)C(═NOR¹¹), CH₂Z′, Z′—CH₂ and CH₂Z′CH₂;

Z′ is selected from C(R¹¹)(OR¹²), O, S, C(═O), C(═NOR¹¹) and NR¹¹;

R⁶ is selected from H, SO₂R^(2a), CO₂R^(2a), C(═O)R^(2a),C(═O)NR^(1c)R^(1d), optionally substituted alkyl, optionally substitutedalkenyl, and optionally substituted alkynyl, wherein said optionalsubstituents are one to about three R⁵ groups; or

alternatively, when Q is NR² and Q′ is NR⁶ or C(R¹⁰)₂, R² and R⁶ or oneof R¹⁰ are joined together to form:

wherein R⁷ and R⁸ are each independently selected from H, OH, alkyl,alkoxy, optionally substituted arylalkyl, optionally substitutedheteroarylalkyl, (CH₂)_(p)OR¹⁰, (CH₂)_(p)OC(═O)NR¹¹R¹² and(CH₂)_(p)NR¹¹R¹²; or R⁷ and R⁸ together form a linking group of theformula CH₂—X³—CH₂;

X³ is a bond, O, S, or NR¹⁰;

J is selected from a bond, O, CH═CH, S, C(═O), CH(OR¹⁰), N(R¹⁰),N(OR¹⁰), CH(NR¹¹R¹²), C(═O)N(R¹⁷), N(R¹⁷)C(═O), N(S(O)_(y)R⁹),N(S(O)_(y)NR¹¹R¹²), N(C(═O)R¹⁷), C(R¹⁵R¹⁶), N⁺(O⁻)(R¹⁰), CH(OH)CH(OH)and CH(O(C═O)R⁹)CH(OC(═O)R⁹);

J′ is selected from O, S, N(R¹⁰), N⁺(O⁻)(R¹⁰), N(OR¹⁰) and CH₂;

R¹³ is selected from alkyl, aryl and arylalkyl;

R¹⁴ is the residue of an amino acid after the hydroxyl group of thecarboxyl group is removed;

R¹⁵ and R¹⁶ are independently selected from H, OH, C(═O)R¹⁰, O(C═O)R⁹,alkyl-OH, alkoxy and CO₂R¹⁰;

R¹⁷ is selected from H, alkyl, aryl and heteroaryl;

R²² is

X⁵ and X⁶ are independently selected from O, N, S, CHR²⁶, C(OH)R²⁶,C(═O) and CH₂═C;

X⁷ and X⁸ are independently selected from a bond, O, N, S, CHR²⁶,C(OH)R²⁶, C(═O) and CH₂═C;

X⁹ and X¹⁰ are independently selected from a bond, O, N, S, C(═O) andCHR²⁶;

X¹¹ is a bond or alkylene optionally substituted with NR¹¹R¹² or OR³⁰;

R²³ is selected from H, OR²⁷, SR²⁷, R²² and R²⁸;

R²⁴ is selected from R, thioalkyl, and halogen;

R²⁵ is selected from R¹ and OC(═O)NR^(1c)R^(1d);

R²⁶ is selected from H, optionally substituted alkyl and optionallysubstituted alkoxy, wherein

(1) ring G contains 0 to about 3 ring heteroatoms;

(2) any two adjacent hydroxyl groups of ring G can be joined to form adioxolane ring;

(3) any two adjacent ring carbon atoms of ring G can be joined to form afused aryl or heteroaryl ring; with the provisos that:

(a) when X¹¹ is a bond, ring G can be heteroaryl; and

(b) ring G:

(i) contains at least one carbon atom that is saturated;

(ii) does not contain two adjacent ring O atoms;

(iii) contains a maximum of two C(═O) groups;

R²⁷ is selected from H and alkyl;

R²⁸ is selected from aryl, arylalkyl, SO₂R²⁹, CO₂R²⁹, C(═O)R²⁹,optionally substituted alkyl, optionally substituted alkenyl andoptionally substituted alkynyl;

R²⁹ is selected from alkyl, aryl and heteroaryl;

R³⁰ is selected from H, alkyl, acyl and C(═O)NR¹¹R¹²;

m is independently selected from 0, 1, and 2;

p is independently selected from 1, 2, 3, and 4;

r is independently selected from 0, 1, and 2;

y is independently selected from 0, 1 and 2; and

z is selected from 0, 1, 2, 3 and 4;

with the provisos that at least one of Y and Q′ is a direct bond, when Yis a direct bond, Q′ is other than a direct bond, when Q′ is a directbond, Y is other than a direct bond, and when rings B and F are phenyl,G-X-W is CH₂NHC(═O), Y is a direct bond, Q is NR² and Q′ is NR⁶ where R⁶is joined with R² to form

then R³ is other than CH₂SCH₂CH₃.

As used herein, the term “sudden sensorineural hearing loss” refers tohearing loss developed as a result of idiopathic factors.

The present invention also provides methods for preserving function ofsensory hair cells prior to or subsequent to trauma in a subjectcomprising administering to said subject an effective amount of a fusedpyrrolocarbazole of Formula I having the formula:

or a stereoisomer or pharmaceutically acceptable salt form thereof,wherein:

ring D is selected from phenyl and cyclohexene with double bond a-b;

ring B and ring F are independently selected from:

(a) a 6-membered carbocyclic ring in which from 1 to 3 carbon atoms maybe replaced by heteroatoms;

(b) a 5-membered carbocyclic ring; and

(c) a 5-membered carbocyclic ring in which either:

(1) one carbon atom is replaced with an oxygen, nitrogen, or sulfuratom;

(2) two carbon atoms are replaced with a sulfur and a nitrogen atom, anoxygen and a nitrogen atom, or two nitrogen atoms; or

(3) three carbon atoms are replaced with three nitrogen atoms, oneoxygen and two nitrogen atoms, or one sulfur and two nitrogen atoms;

G-X-W is selected from:

(a) (Z¹Z²)C—N(R¹)—C(Z¹Z²);

(b) CH(R¹)—C(═O)—N(R¹); and

(c) N(R¹)—C(═O)—CH(R¹);

Z¹ and Z², at each occurrence, are independently selected from H, H; H,OR; H, SR; H, N(R)₂; and a group wherein Z¹ and Z² together form amoiety selected from ═O, ═S, and ═NR; with the proviso that at least oneof the pairs Z¹ and Z² forms ═O;

R is independently selected from H, optionally substituted alkyl, OH,alkoxy, OC(═O)R^(1a), OC(═O)NR^(1c)R^(1d), O(CH₂)_(p)NR^(1c)R^(1d),O(CH₂)_(p)OR^(1b), optionally substituted arylalkyl and optionallysubstituted heteroarylalkyl;

R¹ is independently selected from:

(a) H, optionally substituted alkyl, optionally substituted aryl,optionally substituted arylalkyl, optionally substituted heteroaryl andoptionally substituted heteroarylalkyl;

(b) C(═O)R^(1a);

(c) OR^(1b);

(d) C(═O)NHR^(1b), NR^(1c)R^(1d), (CH₂)_(p)NR^(1c)R^(1d),(CH₂)_(p)OR^(1b), O(CH₂)_(p)OR^(1b) and O(CH₂)_(p)NR^(1c)R^(1d);

R^(1a) is independently selected from optionally substituted alkyl,optionally substituted aryl and optionally substituted heteroaryl;

R^(1b) is independently selected from H and optionally substitutedalkyl;

R^(1c) and R^(1d) are each independently selected from H, optionallysubstituted alkyl and a linking group of the formula (CH₂)₂—X¹—(CH₂)₂;

X¹ is independently selected from O, S and CH₂;

Q is selected from NR², O, S, NR²², CHR²³, X⁴CH(R²³), CH(R²³)X⁴, whereinX⁴ is selected from O, S, CH₂, NR²² and NR²;

R² is selected from H, SO₂R^(2a), CO₂R^(2a), C(═O)R^(2a),C(═O)NR^(2c)R^(2d), optionally substituted alkyl, optionally substitutedalkenyl and optionally substituted alkynyl, wherein said optionalsubstituents are one to about three R⁵ groups;

R^(2a) is independently selected from optionally substituted alkyl,optionally substituted aryl, optionally substituted carbocyclyl,optionally substituted heterocyclyl, OR^(2b), CONH₂, NR^(2c)R^(2d),(CH₂)_(p)NR^(2c)R^(2d) and O(CH₂)_(p)NR^(2c)R^(2d);

R^(2b) is selected from H and optionally substituted alkyl;

R^(2c) and R^(2d) are each independently selected from H and optionallysubstituted alkyl, or together form a linking group of the formula(CH₂)₂—X¹—(CH₂)₂;

R³ and R⁴ are each independently selected from:

(a) H, aryl, heteroaryl, F, Cl, Br, I, CN, CF₃, NO₂, OH, OR⁹,O(CH₂)_(p)NR¹¹R¹², OC(═O)R⁹, OC(═O)NR¹¹R¹², O(CH₂)_(p)OR¹⁰, CH₂OR¹⁰,NR¹¹R¹², NR¹⁰S(═O)₂R⁹ and NR¹⁰C(═O)R⁹;

(b) CH₂OR¹⁴;

(c) NR¹⁰C(═O)NR¹¹R¹², CO₂R¹⁰, C(═O)R⁹, C(═O)NR¹¹R¹², CH═NOR¹⁰, CH═NR¹⁰,(CH₂)_(p)NR¹¹R¹², (CH₂)_(p)NHR¹⁴ and CH═NNR¹¹R¹²;

(d) S(O)_(y)R⁹, (CH₂)_(p)S(O)_(y)R⁹ and CH₂S(O)_(y)R¹⁴;

(e) optionally substituted alkyl, optionally substituted alkenyl andoptionally substituted alkynyl, wherein said optional substituents areone to about three R⁵ groups;

R⁹ is selected from alkyl, (CH₂)_(r)aryl and (CH₂)_(r)heteroaryl;

R¹⁰ is selected from H, alkyl, (CH₂)_(r)aryl and (CH₂)_(r)heteroaryl;

R¹¹ and R¹² are independently selected from H and optionally substitutedalkyl, or together form a linking group of the formula (CH₂)₂—X¹—(CH₂)₂;

R⁵ is selected from aryl, heteroaryl, arylalkoxy, heterocycloalkoxy,hydroxyalkoxy, alkyloxy-alkoxy, hydroxyalkylthio, alkoxy-alkylthio, F,Cl, Br, I, CN, NO₂, OH, OR⁹, X²(CH₂)_(p)NR¹¹R¹²,X²(CH₂)_(p)C(═O)NR¹¹R¹², X²(CH₂)_(p)OC(═O)NR¹¹R¹², X²(CH₂)_(p)CO₂R⁹,X²(CH₂)_(p)S(O)_(y)R⁹, X²(CH₂)_(p)NR¹⁰C(═O)NR¹¹R¹², OC(═O)R⁹,OC(═O)NHR¹⁰, O-tetrahydropyranyl, NR¹¹R¹², NR¹⁰C(═O)R⁹, NR¹⁰CO₂R⁹,NR¹⁰C(═O)NR¹¹R¹², NHC(═NH)NH₂, NR¹⁰S(O)₂R⁹, S(O)_(y)R⁹, CO₂R¹⁰,C(═O)NR¹¹R¹², C(═O)R⁹, CH₂OR¹⁰, CH═NNR¹¹R¹², CH═NOR¹⁰, CH═NR⁹,CH═NNHCH(N═NH)NH₂, S(═O)₂NR¹¹R¹², P(═O)(OR¹⁰)₂, OR¹⁴, and amonosaccharide wherein each hydroxyl group of the monosaccharide isindependently either unsubstituted or is replaced by H, alkyl,alkylcarbonyloxy, or alkoxy;

X² is O, S, or NR¹⁰;

Y is selected from:

(a) a direct bond;

(b) optionally substituted CH₂, CH₂CH₂ or CH₂CH₂CH₂, wherein saidoptional substituents are one to about three R¹⁹ groups; and

(c) CH═CH, CH(OH)—CH(OH), O, S, S(═O), S(═O)₂, C(R¹⁸)₂, C═C(R¹⁹)₂,C(═O), C(═NOR²⁰), C(OR²⁰)R²⁰, C(═O)CH(R¹⁸), CH(R¹⁸)C(═O),C(═NOR²⁰)CH(R¹⁸), CHR²¹C(═NOR²⁰), C(═O)N(R²¹), N(R²¹)C(═O), CH₂Z, ZCH₂and CH₂ZCH₂, where Z is selected from C(R²⁰)₂, O, S, CO₂R²⁰, C(═NOR²⁰)and N(R²⁰);

R¹⁸ is independently selected from H, SO₂R^(18a), CO₂R^(18a),C(═O)R^(18a), C(═O)NR^(18c)R^(18d), optionally substituted alkyl,optionally substituted alkenyl, and optionally substituted alkynyl;

R^(18a) is independently selected from optionally substituted alkyl,optionally substituted aryl, optionally substituted carbocyclyl andoptionally substituted heterocyclyl;

R^(18c) and R^(18d) are each independently selected from H andoptionally substituted alkyl, or together form a linking group of theformula (CH₂)₂—X¹—(CH₂)₂;

R¹⁹ is independently selected from R²⁰, thioalkyl, halogen, optionallysubstituted alkyl, optionally substituted alkenyl and optionallysubstituted alkynyl;

R²⁰ is independently selected from H, alkyl, OH, alkoxy, OC(═O)R^(18a),OC(═O)NR^(18c)R^(18d), OC(═S)NR^(18c)R^(18d), O(CH₂)_(p)NR^(18c)R^(18d),O(CH₂)_(p)OR²¹, optionally substituted arylalkyl, optionally substitutedheterocyclylalkyl and optionally substituted carbocyclyl;

R²¹ is independently selected from H and alkyl;

Q′ is selected from:

(a) a direct bond;

(b) NR⁶;

(c) optionally substituted CH₂, CH₂CH₂ or CH₂CH₂CH₂;

(d) CR²²R²⁴; and

(e) CH═CH, CH(OH)CH(OH), O, S, S(═O), S(═O)₂, C(═O), C(═NOR¹¹),C(OR¹¹)(R¹²), C(═O)CH(R¹³), CH(R¹³)C(═O), C(R¹⁰)₂, C(═NOR¹¹)CH(R¹³),CH(R¹³)C(═NOR¹¹), CH₂Z′, Z′—CH₂ and CH₂Z′CH₂;

Z¹ is selected from C(R¹¹)(OR¹²), O, S, C(═O), C(═NOR¹¹) and NR¹¹;

R⁶ is selected from H, SO₂R^(2a), CO₂R^(2a), C(═O)R^(2a),C(═O)NR^(1c)R^(1d), optionally substituted alkyl, optionally substitutedalkenyl, and optionally substituted alkynyl, wherein said optionalsubstituents are one to about three R⁵ groups; or

alternatively, when Q is NR² and Q′ is NR⁶ or C(R¹⁰)₂, R² and R⁶ or oneof R¹⁰ are joined together to form:

wherein R⁷ and R⁸ are each independently selected from H, OH, alkyl,alkoxy, optionally substituted arylalkyl, optionally substitutedheteroarylalkyl, (CH₂)_(p)OR¹⁰, (CH₂)_(p)OC(═O)NR¹¹R¹² and(CH₂)_(p)NR¹¹R¹²; or R⁷ and R⁸ together form a linking group of theformula CH₂—X³—CH₂;

X³ is a bond, O, S, or NR¹⁰;

J is selected from a bond, O, CH═CH, S, C(═O), CH(OR¹⁰), N(R¹⁰),N(OR¹⁰), CH(NR¹¹R¹²), C(═O)N(R¹⁷), N(R¹⁷)C(═O), N(S(O)_(y)R⁹),N(S(O)_(y)NR¹¹R¹²), N(C(═O)R¹⁷), C(R¹⁵R¹⁶), N⁺(O⁻)(R¹⁰), CH(OH)CH(OH)and CH(O(C═O)R⁹)CH(OC(═O)R⁹);

J′ is selected from O, S, N(R¹⁰), N⁺(O⁻)(R¹⁰), N(OR¹⁰) and CH₂;

R¹³ is selected from alkyl, aryl and arylalkyl;

R¹⁴ is the residue of an amino acid after the hydroxyl group of thecarboxyl group is removed;

R¹⁵ and R¹⁶ are independently selected from H, OH, C(═O)R¹⁰, O(C═O)R⁹,alkyl-OH, alkoxy and CO₂R¹⁰;

R¹⁷ is selected from H, alkyl, aryl and heteroaryl;

R²² is

X⁵ and X⁶ are independently selected from O, N, S, CHR²⁶, C(OH)R²⁶,C(═O) and CH₂═C;

X⁷ and X⁸ are independently selected from a bond, O, N, S, CHR²⁶,C(OH)R²⁶, C(═O) and CH₂═C;

X⁹ and X¹⁰ are independently selected from a bond, O, N, S, C(═O) andCHR²⁶;

X¹¹ is a bond or alkylene optionally substituted with NR¹¹R¹² or OR³⁰;

R²³ is selected from H, OR²⁷, SR²⁷, R²² and R²⁸;

R²⁴ is selected from R, thioalkyl, and halogen;

R²⁵ is selected from R¹ and OC(═O)NR^(1c)CR^(1d);

R²⁶ is selected from H, optionally substituted alkyl and optionallysubstituted alkoxy, wherein

(1) ring G contains 0 to about 3 ring heteroatoms;

(2) any two adjacent hydroxyl groups of ring G can be joined to form adioxolane ring;

(3) any two adjacent ring carbon atoms of ring G can be joined to form afused aryl or heteroaryl ring; with the provisos that:

(a) when X¹¹ is a bond, ring G can be heteroaryl; and

(b) ring G:

(i) contains at least one carbon atom that is saturated;

(ii) does not contain two adjacent ring O atoms;

(iii) contains a maximum of two C(═O) groups;

R²⁷ is selected from H and alkyl;

R²⁸ is selected from aryl, arylalkyl, SO₂R²⁹, CO₂R²⁹, C(═O)R²⁹,optionally substituted alkyl, optionally substituted alkenyl andoptionally substituted alkynyl;

R²⁹ is selected from alkyl, aryl and heteroaryl;

R³⁰ is selected from H, alkyl, acyl and C(═O)NR¹¹R¹²;

m is independently selected from 0, 1, and 2;

p is independently selected from 1, 2, 3, and 4;

r is independently selected from 0, 1, and 2;

y is independently selected from 0, 1 and 2; and

z is selected from 0, 1, 2, 3 and 4;

with the provisos that at least one of Y and Q′ is a direct bond, when Yis a direct bond, Q′ is other than a direct bond, when Q′ is a directbond, Y is other than a direct bond, and when rings B and F are phenyl,G-X-W is CH₂NHC(═O), Y is a direct bond, Q is NR² and Q′ is NR⁶ where R⁶is joined with R² to form

then R³ is other than CH₂SCH₂CH₃.

As used herein, the term “preserving”, in the context of preserving haircell function and the like, refers to maintaining the normal function ofone or more hair cells. As used herein, “preserving” may include, forexample, maintaining at least about 50% of the function of a normal haircell, more preferably at least about 75%, even more preferably at leastabout 80%, even more preferably at least about 85%, even more preferablyat least about 90%, even more preferably at least about 95%, and mostpreferably about 100% of the normal function of the hair cell.

The present invention also provides methods for treating sensory haircells that have been damaged comprising administering to said subject aneffective amount of a fused pyrrolocarbazole of Formula I having theformula:

or a stereoisomer or pharmaceutically acceptable salt form thereof,wherein:

ring D is selected from phenyl and cyclohexene with double bond a-b;

ring B and ring F are independently selected from:

(a) a 6-membered carbocyclic ring in which from 1 to 3 carbon atoms maybe replaced by heteroatoms;

(b) a 5-membered carbocyclic ring; and

(c) a 5-membered carbocyclic ring in which either:

(1) one carbon atom is replaced with an oxygen, nitrogen, or sulfuratom;

(2) two carbon atoms are replaced with a sulfur and a nitrogen atom, anoxygen and a nitrogen atom, or two nitrogen atoms; or

(3) three carbon atoms are replaced with three nitrogen atoms, oneoxygen and two nitrogen atoms, or one sulfur and two nitrogen atoms;

G-X-W is selected from:

(a) (Z¹Z²)C—N(R¹)—C(Z¹Z²);

(b) CH(R¹)—C(═O)—N(R¹); and

(c) N(R¹)—C(═O)—CH(R¹);

Z¹ and Z², at each occurrence, are independently selected from H, H; H,OR; H, SR; H, N(R)₂; and a group wherein Z¹ and Z² together form amoiety selected from ═O, ═S, and ═NR; with the proviso that at least oneof the pairs Z¹ and Z² forms ═O;

R is independently selected from H, optionally substituted alkyl, OH,alkoxy, OC(═O)R^(1a), OC(═O)NR^(1c)R^(1d), O(CH₂)_(p)NR^(1c)R^(1d),O(CH₂)_(p)OR^(1b), optionally substituted arylalkyl and optionallysubstituted heteroarylalkyl;

R¹ is independently selected from:

(a) H, optionally substituted alkyl, optionally substituted aryl,optionally substituted arylalkyl, optionally substituted heteroaryl andoptionally substituted heteroarylalkyl;

(b) C(═O)R^(1a);

(c) OR^(1b);

(d) C(═O)NHR^(1b), NR^(1c)R^(1d), (CH₂)_(p)NR^(1c)R^(1d),(CH₂)_(p)OR^(1b), O(CH₂)_(p)OR^(1b) and O(CH₂)_(p)NR^(1c)R^(1d);

R^(1a) is independently selected from optionally substituted alkyl,optionally substituted aryl and optionally substituted heteroaryl;

R^(1b) is independently selected from H and optionally substitutedalkyl;

R^(1c) and R^(1d) are each independently selected from H, optionallysubstituted alkyl and a linking group of the formula (CH₂)₂—X¹—(CH₂)₂;

X¹ is independently selected from O, S and CH₂;

Q is selected from NR², O, S, NR²², CHR²³, X⁴CH(R²³), CH(R²³)X⁴, whereinX⁴ is selected from O, S, CH₂, NR²² and NR²;

R² is selected from H, SO₂R^(2a), CO₂R^(2a), C(═O)R^(2a),C(═O)NR^(2c)R^(2d), optionally substituted alkyl, optionally substitutedalkenyl and optionally substituted alkynyl, wherein said optionalsubstituents are one to about three R⁵ groups;

R^(2a) is independently selected from optionally substituted alkyl,optionally substituted aryl, optionally substituted carbocyclyl,optionally substituted heterocyclyl, OR^(2b), CONH₂, NR^(2c)R^(2d),(CH₂)_(p)NR^(2c)R^(2d) and O(CH₂)_(p)NR^(2c)R^(2d);

R^(2b) is selected from H and optionally substituted alkyl;

R^(2c) and R^(2d) are each independently selected from H and optionallysubstituted alkyl, or together form a linking group of the formula(CH₂)₂—X¹—(CH₂)₂;

R³ and R⁴ are each independently selected from:

(a) H, aryl, heteroaryl, F, Cl, Br, I, CN, CF₃, NO₂, OH, OR⁹,O(CH₂)_(p)NR¹¹R¹², OC(═O)R⁹, OC(═O)NR¹¹R¹², O(CH₂)_(p)OR¹⁰, CH₂OR¹⁰,NR¹¹R¹², NR¹⁰S(═O)₂R⁹ and NR¹⁰C(═O)R⁹;

(b) CH₂OR¹⁴;

(c) NR¹⁰C(═O)NR¹¹R¹², CO₂R¹⁰, C(═O)R⁹, C(═O)NR¹¹R¹², CH═NOR¹⁰, CH═NR¹⁰,(CH₂)_(p)NR¹¹R¹², (CH₂)_(p)NHR¹⁴ and CH═NNR¹¹R¹²;

(d) S(O)_(y)R⁹, (CH₂)_(p)S(O)_(y)R⁹ and CH₂S(O)_(y)R¹⁴;

(e) optionally substituted alkyl, optionally substituted alkenyl andoptionally substituted alkynyl, wherein said optional substituents areone to about three R⁵ groups;

R⁹ is selected from alkyl, (CH₂)_(r)aryl and (CH₂)_(r)heteroaryl;

R¹⁰ is selected from H, alkyl, (CH₂)_(r)aryl and (CH₂)_(r)heteroaryl;

R¹¹ and R¹² are independently selected from H and optionally substitutedalkyl, or together form a linking group of the formula (CH₂)₂—X¹—(CH₂)₂;

R⁵ is selected from aryl, heteroaryl, arylalkoxy, heterocycloalkoxy,hydroxyalkoxy, alkyloxy-alkoxy, hydroxyalkylthio, alkoxy-alkylthio, F,Cl, Br, I, CN, NO₂, OH, OR⁹, X²(CH₂)_(p)NR¹¹R¹²,X²(CH₂)_(p)C(═O)NR¹¹R¹², X²(CH₂)_(p)OC(═O)NR¹¹R¹², X²(CH₂)_(p)CO₂R⁹,X²(CH₂)_(p)S(O)_(y)R⁹, X²(CH₂)_(p)NR¹⁰C(═O)NR¹¹R¹², OC(═O)R⁹,OC(═O)NHR¹⁰, O-tetrahydropyranyl, NR¹¹R¹², NR¹⁰C(═O)R⁹, NR¹⁰CO₂R⁹,NR¹⁰C(═O)NR¹¹R¹², NHC(═NH)NH₂, NR¹⁰S(O)₂R⁹, S(O)_(y)R⁹, CO₂R¹⁰,C(═O)NR¹¹R¹², C(═O)R⁹, CH₂OR¹⁰, CH═NNR¹¹R¹², CH═NOR¹⁰, CH═NR⁹,CH═NNHCH(N═NH)NH₂, S(═O)₂NR¹¹R¹², P(═O)(OR¹⁰)₂, OR¹⁴, and amonosaccharide wherein each hydroxyl group of the monosaccharide isindependently either unsubstituted or is replaced by H, alkyl,alkylcarbonyloxy, or alkoxy;

X² is O, S, or NR¹⁰;

Y is selected from:

(a) a direct bond;

(b) optionally substituted CH₂, CH₂CH₂ or CH₂CH₂CH₂, wherein saidoptional substituents are one to about three R¹⁹ groups; and

(c) CH═CH, CH(OH)—CH(OH), O, S, S(═O), S(═O)₂, C(R¹⁸)₂, C═C(R¹⁹)₂,C(═O), C(═NOR²⁰), C(OR²⁰)R²⁰, C(═O)CH(R¹⁸), CH(R¹⁸)C(═O),C(═NOR²⁰)CH(R¹⁸), CHR²¹C(═NOR²⁰), C(═O)N(R²¹), N(R²¹)C(═O), CH₂Z, ZCH₂and CH₂ZCH₂, where Z is selected from C(R²⁰)₂, O, S, CO₂R²⁰, C(═NOR²⁰)and N(R²⁰);

R¹⁸ is independently selected from H, SO₂R^(18a), CO₂R^(18a),C(═O)R^(18a), C(═O)NR^(18c)R^(18d), optionally substituted alkyl,optionally substituted alkenyl, and optionally substituted alkynyl;

R^(18a) is independently selected from optionally substituted alkyl,optionally substituted aryl, optionally substituted carbocyclyl andoptionally substituted heterocyclyl;

R^(18c) and R^(18d) are each independently selected from H andoptionally substituted alkyl, or together form a linking group of theformula (CH₂)₂—X¹—(CH₂)₂;

R¹⁹ is independently selected from R²⁰, thioalkyl, halogen, optionallysubstituted alkyl, optionally substituted alkenyl and optionallysubstituted alkynyl;

R²⁰ is independently selected from H, alkyl, OH, alkoxy, OC(═O)R^(18a),OC(═O)NR^(18c)R^(18d), OC(═S)NR^(18c)R^(18d), O(CH₂)_(p)NR^(18c)R^(18d),O(CH₂)_(p)OR²¹, optionally substituted arylalkyl, optionally substitutedheterocyclylalkyl and optionally substituted carbocyclyl;

R²¹ is independently selected from H and alkyl;

Q′ is selected from:

(a) a direct bond;

(b) NR⁶;

(c) optionally substituted CH₂, CH₂CH₂ or CH₂CH₂CH₂;

(d) CR²²R²⁴; and

(e) CH═CH, CH(OH)CH(OH), O, S, S(═O), S(═O)₂, C(═O), C(═NOR¹¹),C(OR¹¹)(R¹²), C(═O)CH(R¹³), CH(R¹³)C(═O), C(R¹⁰)₂, C(═NOR¹¹)CH(R¹³),CH(R¹³)C(═NOR¹¹), CH₂Z′, Z′—CH₂ and CH₂Z′CH₂;

Z′ is selected from C(R¹¹)(OR¹²), O, S, C(═O), C(═NOR¹¹) and NR¹¹;

R⁶ is selected from H, SO₂R^(2a), CO₂R^(2a), C(═O)R^(2a),C(═O)NR^(1c)R^(1d), optionally substituted alkyl, optionally substitutedalkenyl, and optionally substituted alkynyl, wherein said optionalsubstituents are one to about three R⁵ groups; or

alternatively, when Q is NR² and Q′ is NR⁶ or C(R¹⁰)₂, R² and R⁶ or oneof R¹⁰ are joined together to form:

wherein R⁷ and R⁸ are each independently selected from H, OH, alkyl,alkoxy, optionally substituted arylalkyl, optionally substitutedheteroarylalkyl, (CH₂)_(p)OR¹⁰, (CH₂)_(p)OC(═O)NR¹¹R¹² and(CH₂)_(p)NR¹¹R¹²; or R⁷ and R⁸ together form a linking group of theformula CH₂—X³—CH₂;

X³ is a bond, O, S, or NR¹⁰;

J is selected from a bond, O, CH═CH, S, C(═O), CH(OR¹⁰), N(R¹⁰),N(OR¹⁰), CH(NR¹¹R¹²), C(═O)N(R¹⁷), N(R¹⁷)C(═O), N(S(O)_(y)R⁹),N(S(O)_(y)NR¹¹R¹²), N(C(═O)R¹⁷), C(R¹⁵R¹⁶), N⁺(O⁻)(R¹⁰), CH(OH)CH(OH)and CH(O(C═O)R⁹)CH(OC(═O)R⁹);

J′ is selected from O, S, N(R¹⁰), N⁺(O⁻)(R¹⁰), N(OR¹⁰) and CH₂;

R¹³ is selected from alkyl, aryl and arylalkyl;

R¹⁴ is the residue of an amino acid after the hydroxyl group of thecarboxyl group is removed;

R¹⁵ and R¹⁶ are independently selected from H, OH, C(═O)R¹⁰, O(C═O)R⁹,alkyl-OH, alkoxy and CO₂R¹⁰;

R¹⁷ is selected from H, alkyl, aryl and heteroaryl;

R²² is

X⁵ and X⁶ are independently selected from O, N, S, CHR²⁶, C(OH)R²⁶,C(═O) and CH₂═C;

X⁷ and X⁸ are independently selected from a bond, O, N, S, CHR²⁶,C(OH)R²⁶, C(═O) and CH₂═C;

X⁹ and X¹⁰ are independently selected from a bond, O, N, S, C(═O) andCHR²⁶;

X¹¹ is a bond or alkylene optionally substituted with NR¹¹R¹² or OR³⁰;

R²³ is selected from H, OR²⁷, SR²⁷, R²² and R²⁸;

R²⁴ is selected from R, thioalkyl, and halogen;

R²⁵ is selected from R¹ and OC(═O)NR^(1c)R^(1d);

R²⁶ is selected from H, optionally substituted alkyl and optionallysubstituted alkoxy, wherein

(1) ring G contains 0 to about 3 ring heteroatoms;

(2) any two adjacent hydroxyl groups of ring G can be joined to form adioxolane ring;

(3) any two adjacent ring carbon atoms of ring G can be joined to form afused aryl or heteroaryl ring; with the provisos that:

(a) when X¹¹ is a bond, ring G can be heteroaryl; and

(b) ring G:

(i) contains at least one carbon atom that is saturated;

(ii) does not contain two adjacent ring O atoms;

(iii) contains a maximum of two C(═O) groups;

R²⁷ is selected from H and alkyl;

R²⁸ is selected from aryl, arylalkyl, SO₂R²⁹, CO₂R²⁹, C(═O)R²⁹,optionally substituted alkyl, optionally substituted alkenyl andoptionally substituted alkynyl;

R²⁹ is selected from alkyl, aryl and heteroaryl;

R³⁰ is selected from H, alkyl, acyl and C(═O)NR¹¹R¹²;

m is independently selected from 0, 1, and 2;

p is independently selected from 1, 2, 3, and 4;

r is independently selected from 0, 1, and 2;

y is independently selected from 0, 1 and 2; and

z is selected from 0, 1, 2, 3 and 4;

with the provisos that at least one of Y and Q′ is a direct bond, when Yis a direct bond, Q′ is other than a direct bond, when Q′ is a directbond, Y is other than a direct bond, and when rings B and F are phenyl,G—X—W is CH₂NHC(═O), Y is a direct bond, Q is NR² and Q′ is NR⁶ where R⁶is joined with R² to form

then R³ is other than CH₂SCH₂CH₃.

As used herein, the term “treating”, in the context of treating damagedsensory hair cells, refers to the restoration or recovery of the abilityto perceive and/or transduce sensory stimuli of at least some of thehair cells damaged due to a trauma. As used herein, “treating” mayinclude recovering at least about 15%, more preferably at least about25%, even more preferably at least about 50%, even more preferably atleast about 75%, even more preferably at least about 80%, even morepreferably at least about 85%, even more preferably at least about 90%,even more preferably at least about 95%, and most preferably about 100%of the ability of a normal hair cell to perceive and/or transducesensory stimuli of at least some of the hair cells damaged due to atrauma.

As used herein, the term “damaged” refers to one or more sensory hairscell that, due to a trauma, is less able to perceive and/or transduce anexternal stimuli than a normal sensory hair cell.

As used herein, the term “trauma” includes, but is not limited to noise,infection, drug toxicity, aging, disease and idiopathic effects.

The present invention also provides methods for treating sensory haircells that have been damaged comprising administering to said subject aneffective amount of a fused pyrrolocarbazole of Formula I having theformula:

or a stereoisomer or pharmaceutically acceptable salt form thereof,wherein:

ring D is selected from phenyl and cyclohexene with double bond a-b;

ring B and ring F are independently selected from:

(a) a 6-membered carbocyclic ring in which from 1 to 3 carbon atoms maybe replaced by heteroatoms;

(b) a 5-membered carbocyclic ring; and

(c) a 5-membered carbocyclic ring in which either:

(1) one carbon atom is replaced with an oxygen, nitrogen, or sulfuratom;

(2) two carbon atoms are replaced with a sulfur and a nitrogen atom, anoxygen and a nitrogen atom, or two nitrogen atoms; or

(3) three carbon atoms are replaced with three nitrogen atoms, oneoxygen and two nitrogen atoms, or one sulfur and two nitrogen atoms;

G—X—W is selected from:

(a) (Z¹Z²)C—N(R¹)—C(Z¹Z²);

(b) CH(R¹)—C(═O)—N(R¹); and

(c) N(R¹)—C(═O)—CH(R¹);

Z¹ and Z², at each occurrence, are independently selected from H, H; H,OR; H, SR; H, N(R)₂; and a group wherein Z¹ and Z² together form amoiety selected from ═O, ═S, and ═NR; with the proviso that at least oneof the pairs Z¹ and Z² forms ═O;

R is independently selected from H, optionally substituted alkyl, OH,alkoxy, OC(═O)R^(1a), OC(═O)NR^(1c)R^(1d), O(CH₂)_(p)NR^(1c)R^(1d),O(CH₂)_(p)OR^(1b), optionally substituted arylalkyl and optionallysubstituted heteroarylalkyl;

R¹ is independently selected from:

(a) H, optionally substituted alkyl, optionally substituted aryl,optionally substituted arylalkyl, optionally substituted heteroaryl andoptionally substituted heteroarylalkyl;

(b) C(═O)R^(1a);

(c) OR^(1b);

(d) C(═O)NHR^(1b),NR^(1c)R^(1d), (CH₂)_(p)NR^(1c)R^(1d),(CH₂)_(p)OR^(1b), O(CH₂)_(p)OR_(1b) and O(CH₂)_(p)NR^(1c)R^(1d);

R^(1a) is independently selected from optionally substituted alkyl,optionally substituted aryl and optionally substituted heteroaryl;

R_(1b) is independently selected from H and optionally substitutedalkyl;

R^(1c) and R^(1d) are each independently selected from H, optionallysubstituted alkyl and a linking group of the formula (CH₂)₂—X¹—(CH₂)₂;

X¹ is independently selected from O, S and CH₂;

Q is selected from NR², O, S, NR²², CHR²³, X⁴CH(R²³), CH(R²³)X⁴, whereinX⁴ is selected from O, S, CH₂, NR²² and NR²;

R² is selected from H, SO₂R^(2a), CO₂R^(2a), C(═O)R^(2a),C(═O)NR^(2c)R^(2d), optionally substituted alkyl, optionally substitutedalkenyl and optionally substituted alkynyl, wherein said optionalsubstituents are one to about three R′ groups;

R^(2a), is independently selected from optionally substituted alkyl,optionally substituted aryl, optionally substituted carbocyclyl,optionally substituted heterocyclyl, OR^(2b), CONH₂, NR^(2c)R^(2d),(CH₂)_(p)NR^(2c)R^(2d) and O(CH₂)_(p)NR^(2c)R^(2d);

R^(2b) is selected from H and optionally substituted alkyl;

R^(2c) and R^(2d) are each independently selected from H and optionallysubstituted alkyl, or together form a linking group of the formula(CH₂)₂—X¹—(CH₂)₂;

R³ and R⁴ are each independently selected from:

(a) H, aryl, heteroaryl, F, Cl, Br, I, CN, CF₃, NO₂, OH, OR⁹,O(CH₂)_(p)NR¹¹R¹², OC(═O)R⁹, OC(═O)NR¹¹R¹², O(CH₂)_(p)OR¹⁰, CH₂OR¹⁰,NR¹¹R¹², NR¹⁰S(═O)₂R⁹ and NR¹⁰C(═O)R⁹;

(b) CH₂OR¹⁴;

(c) NR¹⁰C(═O)NR¹¹R¹², CO₂R¹⁰, C(═O)R⁹, C(═O)NR¹¹R¹², CH═NOR¹⁰, CH═NR¹⁰,(CH₂)_(p)NR¹¹R¹², (CH₂)_(p)NHR¹⁴ and CH═NNR¹¹R¹²;

(d) S(O)_(y)R⁹, (CH₂)_(p)S(O)_(y)R⁹ and CH₂S(O)_(y)R¹⁴;

(e) optionally substituted alkyl, optionally substituted alkenyl andoptionally substituted alkynyl, wherein said optional substituents areone to about three R⁵ groups;

R⁹ is selected from alkyl, (CH₂)_(r)aryl and (CH₂)_(r)heteroaryl;

R¹⁰ is selected from H, alkyl, (CH₂)_(r)aryl and (CH₂)_(r)heteroaryl;

R¹¹ and R¹² are independently selected from H and optionally substitutedalkyl, or together form a linking group of the formula (CH₂)₂—X¹—(CH₂)₂;

R⁵ is selected from aryl, heteroaryl, arylalkoxy, heterocycloalkoxy,hydroxyalkoxy, alkyloxy-alkoxy, hydroxyalkylthio, alkoxy-alkylthio, F,Cl, Br, I, CN, NO₂, OH, OR⁹, X²(CH₂)_(p)NR¹¹R¹²,X²(CH₂)_(p)C(═O)NR¹¹R¹², X²(CH₂)_(p)OC(═O)NR¹¹R¹², X²(CH₂)_(p)CO₂R⁹,X²(CH₂)_(p)S(O)_(y)R⁹, X²(CH₂)_(p)NR¹⁰C(═O)NR¹¹R¹², OC(═O)R⁹,OC(═O)NHR¹⁰, O-tetrahydropyranyl, NR¹¹R¹², NR¹⁰C(═O)R⁹, NR¹⁰CO₂R⁹,NR¹⁰C(═O)NR¹¹R¹², NHC(═NH)NH₂, NR¹⁰S(O)₂R⁹, S(O)_(y)R⁹, CO₂R¹⁰,C(═O)NR¹¹R¹², C(═O)R⁹, CH₂OR¹⁰, CH═NNR¹¹R¹², CH═NOR¹⁰, CH═NR⁹,CH═NNHCH(N═NH)NH₂, S(═O)₂NR¹¹R¹², P(═O)(OR¹⁰)₂, OR¹⁴, and amonosaccharide wherein each hydroxyl group of the monosaccharide isindependently either unsubstituted or is replaced by H, alkyl,alkylcarbonyloxy, or alkoxy;

X² is O, S, or NR¹⁰;

Y is selected from:

(a) a direct bond;

(b) optionally substituted CH₂, CH₂CH₂ or CH₂CH₂CH₂, wherein saidoptional substituents are one to about three R¹⁹ groups; and

(c) CH═CH, CH(OH)—CH(OH), O, S, S(═O), S(═O)₂, C(R¹⁸)₂, C═C(R¹⁹)₂,C(═O), C(═NOR²⁰), C(OR²⁰)R²⁰, C(═O)CH(R¹⁸), CH(R¹⁸)C(═O),C(═NOR²⁰)CH(R¹⁸), CHR²¹C(═NOR²⁰), C(═O)N(R²¹), N(R²¹)C(═O), CH₂Z, ZCH₂and CH₂ZCH₂, where Z is selected from C(R²⁰)₂, O, S, CO₂R²⁰, C(═NOR²⁰)and N(R²⁰);

R¹⁸ is independently selected from H, SO₂R^(18a),CO₂R^(18a),C(═O)R^(18a), C(═O)NR^(18c)R^(18d), optionally substituted alkyl,optionally substituted alkenyl, and optionally substituted alkynyl;

R^(18a) is independently selected from optionally substituted alkyl,optionally substituted aryl, optionally substituted carbocyclyl andoptionally substituted heterocyclyl;

R^(18c) and R^(18d) are each independently selected from H andoptionally substituted alkyl, or together form a linking group of theformula (CH₂)₂—X¹—(CH₂)₂;

R¹⁹ is independently selected from R²⁰, thioalkyl, halogen, optionallysubstituted alkyl, optionally substituted alkenyl and optionallysubstituted alkynyl;

R²⁰ is independently selected from H, alkyl, OH, alkoxy, OC(═O)R^(18a),OC(═O)NR^(18c)R^(18d), OC(═S)NR^(18c)R^(18d), O(CH₂)_(p)NR^(18c)R^(18d),O(CH₂)_(p)OR²¹, optionally substituted arylalkyl, optionally substitutedheterocyclylalkyl and optionally substituted carbocyclyl;

R²¹ is independently selected from H and alkyl;

Q′ is selected from:

(a) a direct bond;

(b) NR⁶;

(c) optionally substituted CH₂, CH₂CH₂ or CH₂CH₂CH₂;

(d) CR²²R²⁴; and

(e) CH═CH, CH(OH)CH(OH), O, S, S(═O), S(═O)₂, C(═O), C(═NOR¹¹),C(OR¹¹)(R¹²), C(═O)CH(R¹³), CH(R¹³)C(═O), C(R¹⁰)₂, C(═NOR¹¹)CH(R¹³),CH(R¹³)C(═NOR¹¹), CH₂Z′, Z′—CH₂ and CH₂Z′CH₂;

Z′ is selected from C(R¹¹)(OR¹²), O, S, C(═O), C(═NOR¹¹) and NR¹¹;

R⁶ is selected from H, SO₂R^(2a), CO₂R^(2a), C(═O)R^(2a),C(═O)NR^(1c)R^(1d), optionally substituted alkyl, optionally substitutedalkenyl, and optionally substituted alkynyl, wherein said optionalsubstituents are one to about three R⁵ groups; or

alternatively, when Q is NR² and Q′ is NR⁶ or C(R¹⁰)₂, R² and R⁶ or oneof R¹⁰ are joined together to form:

wherein R⁷ and R⁸ are each independently selected from H, OH, alkyl,alkoxy, optionally substituted arylalkyl, optionally substitutedheteroarylalkyl, (CH₂)_(p)OR¹⁰, (CH₂)_(p)OC(═O)NR¹¹R¹² and(CH₂)_(p)NR¹¹R¹²; or R⁷ and R⁸ together form a linking group of theformula CH₂—X³—CH₂;

X³ is a bond, O, S, or NR¹⁰;

J is selected from a bond, 0, CH═CH, S, C(═O), CH(OR¹⁰), N(R¹⁰),N(OR¹⁰), CH(NR¹¹R¹²), C(═O)N(R¹⁷), N(R¹⁷)C(═O), N(S(O)_(y)R⁹),N(S(O)_(y)NR¹¹R¹²), N(C(═O)R¹⁷), C(R¹⁵R¹⁶), N⁺(O⁻)(R¹⁰), CH(OH)CH(OH)and CH(O(C═O)R⁹)CH(OC(═O)R⁹);

J′ is selected from O, S, N(R¹⁰), N⁺(O⁻)(R¹⁰), N(OR¹⁰) and CH₂;

R¹³ is selected from alkyl, aryl and arylalkyl;

R¹⁴ is the residue of an amino acid after the hydroxyl group of thecarboxyl group is removed;

R¹⁵ and R¹⁶ are independently selected from H, OH, C(═O)R¹⁰, O(C═O)R⁹,alkyl-OH, alkoxy and CO₂R¹⁰;

R¹⁷ is selected from H, alkyl, aryl and heteroaryl;

R²² is

X⁵ and X⁶ are independently selected from O, N, S, CHR²⁶, C(OH)R²⁶,C(═O) and CH₂═C;

X⁷ and X⁸ are independently selected from a bond, O, N, S, CHR²⁶,C(OH)R²⁶, C(═O) and CH₂═C;

X⁹ and X¹⁰ are independently selected from a bond, O, N, S, C(═O) andCHR²⁶;

X¹¹ is a bond or alkylene optionally substituted with NR¹¹R¹² or OR³⁰;

R²³ is selected from H, OR²⁷, SR²⁷, R²² and R²⁸;

R²⁴ is selected from R, thioalkyl, and halogen;

R²⁵ is selected from R¹ and OC(═O)NR^(1c)CR^(1d);

R²⁶ is selected from H, optionally substituted alkyl and optionallysubstituted alkoxy, wherein

(1) ring G contains 0 to about 3 ring heteroatoms;

(2) any two adjacent hydroxyl groups of ring G can be joined to form adioxolane ring;

(3) any two adjacent ring carbon atoms of ring G can be joined to form afused aryl or heteroaryl ring; with the provisos that:

(a) when X¹¹ is a bond, ring G can be heteroaryl; and

(b) ring G:

(i) contains at least one carbon atom that is saturated;

(ii) does not contain two adjacent ring O atoms;

(iii) contains a maximum of two C(═O) groups;

R²⁷ is selected from H and alkyl;

R²⁸ is selected from aryl, arylalkyl, SO₂R²⁹, CO₂R²⁹, C(═O)R²⁹,optionally substituted alkyl, optionally substituted alkenyl andoptionally substituted alkynyl;

R²⁹ is selected from alkyl, aryl and heteroaryl;

R³⁰ is selected from H, alkyl, acyl and C(═O)NR¹¹R¹²;

m is independently selected from 0, 1, and 2;

p is independently selected from 1, 2, 3, and 4;

r is independently selected from 0, 1, and 2;

y is independently selected from 0, 1 and 2; and

z is selected from 0, 1, 2, 3 and 4;

with the provisos that at least one of Y and Q′ is a direct bond, when Yis a direct bond, Q′ is other than a direct bond, when Q′ is a directbond, Y is other than a direct bond, and when rings B and F are phenyl,G—X—W is CH₂NHC(═O), Y is a direct bond, Q is NR² and Q′ is NR⁶ where R⁶is joined with R² to form

then R³ is other than CH₂SCH₂CH₃.

The present invention also provides methods for preventing death ofcochlear neurons in a subject comprising administering to said subjectan effective amount of a fused pyrrolocarbazole of Formula I having theformula:

or a stereoisomer or pharmaceutically acceptable salt form thereof,wherein:

ring D is selected from phenyl and cyclohexene with double bond a-b;ring B and ring F are independently selected from:

(a) a 6-membered carbocyclic ring in which from 1 to 3 carbon atoms maybe replaced by heteroatoms;

(b) a 5-membered carbocyclic ring; and

(c) a 5-membered carbocyclic ring in which either:

(1) one carbon atom is replaced with an oxygen, nitrogen, or sulfuratom;

(2) two carbon atoms are replaced with a sulfur and a nitrogen atom, anoxygen and a nitrogen atom, or two nitrogen atoms; or

(3) three carbon atoms are replaced with three nitrogen atoms, oneoxygen and two nitrogen atoms, or one sulfur and two nitrogen atoms;

G—X—W is selected from:

(a) (Z¹Z²)C—N(R¹)—C(Z¹Z²);

(b) CH(R¹)—C(═O)—N(R¹); and

(c) N(R¹)—C(═O)—CH(R¹);

Z¹ and Z², at each occurrence, are independently selected from H, H; H,OR; H, SR; H, N(R)₂; and a group wherein Z¹ and Z² together form amoiety selected from ═O, ═S, and ═NR; with the proviso that at least oneof the pairs Z¹ and Z² forms ═O;

R is independently selected from H, optionally substituted alkyl, OH,alkoxy, OC(═O)R^(1a), OC(═O)NR^(1c)R^(1d), O(CH₂)_(p)NR^(1c)R^(1d),O(CH₂)_(p)OR^(1b), optionally substituted arylalkyl and optionallysubstituted heteroarylalkyl;

R¹ is independently selected from:

(a) H, optionally substituted alkyl, optionally substituted aryl,optionally substituted arylalkyl, optionally substituted heteroaryl andoptionally substituted heteroarylalkyl;

(b) C(═O)R^(1a);

(c) OR^(1b);

(d) C(═O)NHR^(1b), NR^(1c)R^(1d), (CH₂)_(p)NR^(1c)R^(1d),(CH₂)_(p)OR^(1b), O(CH₂)_(p)OR_(1b) and O(CH₂)_(p)NR^(1c)R^(1d);

R^(1a) is independently selected from optionally substituted alkyl,optionally substituted aryl and optionally substituted heteroaryl;

R_(1b) is independently selected from H and optionally substitutedalkyl;

R^(1c) and R^(1d) are each independently selected from H, optionallysubstituted alkyl and a linking group of the formula (CH₂)₂—X¹—(CH₂)₂;

X¹ is independently selected from O, S and CH₂;

Q is selected from NR², O, S, NR²², CHR²³, X⁴CH(R²³), CH(R²³)X⁴, whereinX⁴ is selected from O, S, CH₂, NR²² and NR²;

R² is selected from H, SO₂R^(2a), CO₂R^(2a), C(═O)R^(2a),C(═O)NR^(2c)R^(2d) optionally substituted alkyl, optionally substitutedalkenyl and optionally substituted alkynyl, wherein said optionalsubstituents are one to about three R⁵ groups;

R^(2a) is independently selected from optionally substituted alkyl,optionally substituted aryl, optionally substituted carbocyclyl,optionally substituted heterocyclyl, OR^(2b), CONH₂, NR^(2c)R^(2d),(CH₂)_(p)NR^(2c)R^(2d) and O(CH₂)_(p)NR^(2c)R^(2d);

R^(2b) is selected from H and optionally substituted alkyl;

R^(2c) and R^(2d) are each independently selected from H and optionallysubstituted alkyl, or together form a linking group of the formula(CH₂)₂—X¹—(CH₂)₂;

R³ and R⁴ are each independently selected from:

(a) H, aryl, heteroaryl, F, Cl, Br, I, CN, CF₃, NO₂, OH, OR⁹,O(CH₂)_(p)NR¹¹R¹², OC(═O)R⁹, OC(═O)NR¹¹R¹², O(CH₂)_(p)OR¹⁰, CH₂OR¹⁰,NR¹¹R¹², NR¹⁰S(═O)₂R⁹ and NR¹⁰C(═O)R⁹;

(b) CH₂OR¹⁴;

(c) NR¹⁰C(═O)NR¹¹R¹², CO₂R¹⁰, C(═O)R⁹, C(═O)NR¹¹R¹², CH═NOR¹⁰, CH═NR¹⁰,(CH₂)_(p)NR¹¹R¹², (CH₂)_(p)NHR¹⁴ and CH═NNR¹¹R¹²;

(d) S(O)_(y)R⁹, (CH₂)_(p)S(O)_(y)R⁹ and CH₂S(O)_(y)R¹⁴;

(e) optionally substituted alkyl, optionally substituted alkenyl andoptionally substituted alkynyl, wherein said optional substituents areone to about three R⁵ groups;

R⁹ is selected from alkyl, (CH₂)_(r)aryl and (CH₂)_(r)heteroaryl;

R¹⁰ is selected from H, alkyl, (CH₂)_(r)aryl and (CH₂)_(r)heteroaryl;

R¹¹ and R¹² are independently selected from H and optionally substitutedalkyl, or together form a linking group of the formula (CH₂)₂—X¹—(CH₂)₂;

R⁵ is selected from aryl, heteroaryl, arylalkoxy, heterocycloalkoxy,hydroxyalkoxy, alkyloxy-alkoxy, hydroxyalkylthio, alkoxy-alkylthio, F,Cl, Br, I, CN, NO₂, OH, OR⁹, X²(CH₂)_(p)NR¹¹R¹²,X²(CH₂)_(p)C(═O)NR¹¹R¹², X²(CH₂)_(p)OC(═O)NR¹¹R¹², X²(CH₂)_(p)CO₂R⁹,X²(CH₂)_(p)S(O)_(y)R⁹, X²(CH₂)_(p)NR¹⁰C(═O)NR¹¹R¹², OC(═O)R⁹,OC(═O)NHR¹⁰, O-tetrahydropyranyl, NR¹¹R¹², NR¹⁰C(═O)R⁹, NR¹⁰CO₂R⁹,NR¹⁰C(═O)NR¹¹R¹², NHC(═NH)NH₂, NR¹⁰S(O)₂R⁹, S(O)_(y)R⁹, CO₂R¹⁰,C(═O)NR¹¹R¹², C(═O)R⁹, CH₂OR¹⁰, CH═NNR¹¹R¹², CH═NOR¹⁰, CH═NR⁹,CH═NNHCH(N═NH)NH₂, S(═O)₂NR¹¹R¹², P(═O)(OR¹⁰)₂, OR¹⁴, and amonosaccharide wherein each hydroxyl group of the monosaccharide isindependently either unsubstituted or is replaced by H, alkyl,alkylcarbonyloxy, or alkoxy;

X² is O, S, or NR¹⁰;

Y is selected from:

(a) a direct bond;

(b) optionally substituted CH₂, CH₂CH₂ or CH₂CH₂CH₂, wherein saidoptional substituents are one to about three R¹⁹ groups; and

(c) CH═CH, CH(OH)—CH(OH), O, S, S(═O), S(═O)₂, C(R¹⁸)₂, C═C(R¹⁹)₂,C(═O), C(═NOR²⁰), C(OR²⁰)R²⁰, C(═O)CH(R¹⁸), CH(R¹⁸)C(═O),C(═NOR²⁰)CH(R¹⁸), CHR21C(═NOR²⁰), C(═O)N(R²¹), N(R²¹)C(═O), CH₂Z, ZCH₂and CH₂ZCH₂, where Z is selected from C (R²⁰)₂, O, S, CO₂R²⁰, C(═NOR²⁰)and N(R²⁰);

R¹⁸ is independently selected from H, SO₂R^(18a), CO₂R^(18a),C(═O)R^(18a), C(═O)NR¹⁸CR^(18d), optionally substituted alkyl,optionally substituted alkenyl, and optionally substituted alkynyl;

R^(18a) is independently selected from optionally substituted alkyl,optionally substituted aryl, optionally substituted carbocyclyl andoptionally substituted heterocyclyl;

R^(18c) and R^(18d) are each independently selected from H andoptionally substituted alkyl, or together form a linking group of theformula (CH₂)₂—X¹—(CH₂)₂;

R¹⁹ is independently selected from R²⁰, thioalkyl, halogen, optionallysubstituted alkyl, optionally substituted alkenyl and optionallysubstituted alkynyl;

R²⁰ is independently selected from H, alkyl, OH, alkoxy, OC(═O)R^(18a),OC(═O)NR^(18c)R^(18d), OC(═S)NR^(18c)R^(18d),O(CH₂)_(p)NR^(18c)R^(18d)O(CH₂)_(p)OR²¹, optionally substitutedarylalkyl, optionally substituted heterocyclylalkyl and optionallysubstituted carbocyclyl;

R²¹ is independently selected from H and alkyl;

Q′ is selected from:

(a) a direct bond;

(b) NR⁶;

(c) optionally substituted CH₂, CH₂CH₂ or CH₂CH₂CH₂;

(d) CR²²R²⁴; and

(e) CH═CH, CH(OH)CH(OH), O, S, S(═O), S(═O)₂, C(═O), C(═NOR¹¹),C(OR¹¹)(R¹²), C(═O)CH(R¹³), CH(R¹³)C(═O), C(R¹⁰)₂, C(═NOR¹¹)CH(R¹³),CH(R¹³)C(═NOR¹¹), CH₂Z′, Z′-CH₂ and CH₂Z′CH₂;

Z¹ is selected from C(R¹¹)(OR¹²), O, S, C(═O), C(═NOR¹¹) and NR¹¹;

R⁶ is selected from H, SO₂R^(2a), CO₂R^(2a),C(═O)R^(2a),C(═O)NR^(1c)R^(1d), optionally substituted alkyl, optionally substitutedalkenyl, and optionally substituted alkynyl, wherein said optionalsubstituents are one to about three R⁵ groups; or

alternatively, when Q is NR² and Q′ is NR⁶ or C(R¹⁰)₂, R² and R⁶ or oneof R¹⁰ are joined together to form:

wherein R⁷ and R⁸ are each independently selected from H, OH, alkyl,alkoxy, optionally substituted arylalkyl, optionally substitutedheteroarylalkyl, (CH₂)_(p)OR¹⁰, (CH₂)_(p)OC(═O)NR¹¹R¹² and(CH₂)_(p)NR¹¹R¹²; or R⁷ and R⁸ together form a linking group of theformula CH₂—X³—CH₂;

X³ is a bond, O, S, or NR¹⁰;

J is selected from a bond, O, CH═CH, S, C(═O), CH(OR¹⁰), N(R¹⁰),N(OR¹⁰), CH(NR¹¹R¹²), C(═O)N(R¹⁷), N(R¹⁷)C(═O), N(S(O)_(y)R⁹),N(S(O)_(y)NR¹¹R¹²), N(C(═O)R¹⁷), C(R¹⁵R¹⁶), N⁺(O⁻)(R¹⁰), CH(OH)CH(OH)and CH(O(C═O)R⁹)CH(OC(═O)R⁹);

J′ is selected from O, S, N(R¹⁰), N⁺(O⁻)(R¹⁰), N(OR¹⁰) and CH₂;

R¹³ is selected from alkyl, aryl and arylalkyl;

R¹⁴ is the residue of an amino acid after the hydroxyl group of thecarboxyl group is removed;

R¹⁵ and R¹⁶ are independently selected from H, OH, C(═O)R¹⁰, O(C═O)R⁹,alkyl-OH, alkoxy and CO₂R¹⁰;

R¹⁷ is selected from H, alkyl, aryl and heteroaryl;

R²² is

X⁵ and X⁶ are independently selected from O, N, S, CHR²⁶, C(OH)R²⁶,C(═O) and CH₂═C;

X⁷ and X⁸ are independently selected from a bond, O, N, S, CHR²⁶,C(OH)R²⁶, C(═O) and CH₂═C;

X⁹ and X¹⁰ are independently selected from a bond, O, N, S, C(═O) andCHR²⁶;

X¹¹ is a bond or alkylene optionally substituted with NR¹¹R¹² or OR³⁰;

R²³ is selected from H, OR²⁷, SR²⁷, R²² and R²⁸;

R²⁴ is selected from R, thioalkyl, and halogen;

R²⁵ is selected from R¹ and OC(═O)NR^(1c)R^(1d);

R²⁶ is selected from H, optionally substituted alkyl and optionallysubstituted alkoxy, wherein

(1) ring G contains 0 to about 3 ring heteroatoms;

(2) any two adjacent hydroxyl groups of ring G can be joined to form adioxolane ring;

(3) any two adjacent ring carbon atoms of ring G can be joined to form afused aryl or heteroaryl ring; with the provisos that:

(a) when X¹¹ is a bond, ring G can be heteroaryl; and

(b) ring G:

(i) contains at least one carbon atom that is saturated;

(ii) does not contain two adjacent ring O atoms;

(iii) contains a maximum of two C(═O) groups;

R²⁷ is selected from H and alkyl;

R²⁸ is selected from aryl, arylalkyl, SO₂R²⁹, CO₂R²⁹, C(═O)R²⁹,optionally substituted alkyl, optionally substituted alkenyl andoptionally substituted alkynyl;

R²⁹ is selected from alkyl, aryl and heteroaryl;

R³⁰ is selected from H, alkyl, acyl and C(═O)NR¹¹R¹²;

m is independently selected from 0, 1, and 2;

p is independently selected from 1, 2, 3, and 4;

r is independently selected from 0, 1, and 2;

y is independently selected from 0, 1 and 2; and

z is selected from 0, 1, 2, 3 and 4;

with the provisos that at least one of Y and Q′ is a direct bond, when Yis a direct bond, Q′ is other than a direct bond, when Q′ is a directbond, Y is other than a direct bond, and when rings B and F are phenyl,G—X—W is CH₂NHC(═O), Y is a direct bond, Q is NR² and Q′ is NR⁶ where R⁶is joined with R² to form

then R³ is other than CH₂SCH₂CH₃.

As used herein, the term “death of cochlear neurons” refers to acessation of the ability of a neuron to transmit impulses from its inputsource to a final destination, as compared with a normally functioningneuron.

The present invention also provides methods for preventing death ofcochlear neurons in a subject comprising administering to said subjectan effective amount of a fused pyrrolocarbazole of Formula 1 having theformula:

or a stereoisomer or pharmaceutically acceptable salt form thereof,wherein:

ring D is selected from phenyl and cyclohexene with double bond a-b;

ring B and ring F are independently selected from:

(a) a 6-membered carbocyclic ring in which from 1 to 3 carbon atoms maybe replaced by heteroatoms;

(b) a 5-membered carbocyclic ring; and

(c) a 5-membered carbocyclic ring in which either:

(1) one carbon atom is replaced with an oxygen, nitrogen, or sulfuratom;

(2) two carbon atoms are replaced with a sulfur and a nitrogen atom, anoxygen and a nitrogen atom, or two nitrogen atoms; or

(3) three carbon atoms are replaced with three nitrogen atoms, oneoxygen and two nitrogen atoms, or one sulfur and two nitrogen atoms;

G—X—W is selected from:

(a) (Z¹Z²)C—N(R¹)—C(Z¹Z²);

(b) CH(R¹)—C(═O)—N(R¹); and

(c) N(R¹)—C(═O)—CH(R¹);

Z¹ and Z², at each occurrence, are independently selected from H, H; H,OR; H, SR; H, N(R)₂; and a group wherein Z¹ and Z² together form amoiety selected from ═O, ═S, and ═NR; with the proviso that at least oneof the pairs Z¹ and Z² forms ═O;

R is independently selected from H, optionally substituted alkyl, OH,alkoxy, OC(═O)R^(1a), OC(═O)NR^(1c)R^(1d), O(CH₂)_(p)NR^(1c)R^(1d),O(CH₂)_(p)OR optionally substituted arylalkyl and optionally substitutedheteroarylalkyl;

R¹ is independently selected from:

(a) H, optionally substituted alkyl, optionally substituted aryl,optionally substituted arylalkyl, optionally substituted heteroaryl andoptionally substituted heteroarylalkyl;

(b) C(═O)R^(1a);

(c) OR;

(d) C(═O)NHR^(1b), NR^(1c)R^(1d), (CH₂)_(p)NR^(1c)R^(1d),(CH₂)_(p)OR^(1b), O(CH₂)_(p)OR_(1b) and O(CH₂)_(p)NR^(1c)R^(1d);

R^(1a) is independently selected from optionally substituted alkyl,optionally substituted aryl and optionally substituted heteroaryl;

R^(1b) is independently selected from H and optionally substitutedalkyl;

R^(1c) and R^(1d) are each independently selected from H, optionallysubstituted alkyl and a linking group of the formula (CH₂)₂—X¹—(CH₂)₂;

X¹ is independently selected from O, S and CH₂;

Q is selected from NR², O, S, NR²², CHR²³, X⁴CH(R²³), CH(R²³)X⁴, whereinX⁴ is selected from O, S, CH₂, NR²² and NR²;

R² is selected from H, SO₂R^(2a), CO₂R^(2a), C(═O)R^(2a),C(═O)NR^(2c)R^(2d), optionally substituted alkyl, optionally substitutedalkenyl and optionally substituted alkynyl, wherein said optionalsubstituents are one to about three R⁵ groups;

R^(2a) is independently selected from optionally substituted alkyl,optionally substituted aryl, optionally substituted carbocyclyl,optionally substituted heterocyclyl, OR^(2b), CONH₂, NR^(2c)R^(2d),(CH₂)_(p)NR^(2c)R^(2d) and O(CH₂)_(p)NR^(2c)R^(2d);

R^(2b) is selected from H and optionally substituted alkyl;

R^(2c) and R^(2d) are each independently selected from H and optionallysubstituted alkyl, or together form a linking group of the formula(CH₂)₂—X¹—(CH₂)₂;

R³ and R⁴ are each independently selected from:

(a) H, aryl, heteroaryl, F, Cl, Br, I, CN, CF₃, NO₂, OH, OR⁹,O(CH₂)_(p)NR¹¹R¹², OC(═O)R⁹, OC(═O)NR¹¹R¹², O(CH₂)_(p)OR¹⁰, CH₂OR¹⁰,NR¹¹R¹², NR¹⁰S(═O)₂R⁹ and NR¹⁰C(═O)R⁹;

(b) CH₂OR¹⁴;

(c) NR¹⁰C(═O)NR¹¹R¹², CO₂R¹⁰, C(═O)R⁹, C(═O)NR¹¹R¹², CH═NOR¹⁰, CH═NR¹⁰,(CH₂)_(p)NR¹¹R¹², (CH₂)_(p)NHR¹⁴ and CH═NNR¹¹R¹²;

(d) S(O)_(y)R⁹, (CH₂)_(p)S(O)_(y)R⁹ and CH₂S(O)_(y)R¹⁴;

(e) optionally substituted alkyl, optionally substituted alkenyl andoptionally substituted alkynyl, wherein said optional substituents areone to about three R⁵ groups;

R⁹ is selected from alkyl, (CH₂)_(r)aryl and (CH₂)_(r)heteroaryl;

R¹⁰ is selected from H, alkyl, (CH₂)_(r)aryl and (CH₂)_(r)heteroaryl;

R¹¹ and R¹² are independently selected from H and optionally substitutedalkyl, or together form a linking group of the formula (CH₂)₂—X¹—(CH₂)₂;

R⁵ is selected from aryl, heteroaryl, arylalkoxy, heterocycloalkoxy,hydroxyalkoxy, alkyloxy-alkoxy, hydroxyalkylthio, alkoxy-alkylthio, F,Cl, Br, I, CN, NO₂, OH, OR⁹, X²(CH₂)_(p)NR¹¹R¹²,X²(CH₂)_(p)C(═O)NR¹¹R¹², X²(CH₂)_(p)OC(═O)NR¹¹R¹², X²(CH₂)_(p)CO₂R⁹,X²(CH₂)_(p)S(O)_(y)R⁹, X²(CH₂)_(p)NR¹⁰C(═O)NR¹¹R¹², OC(═O)R⁹,OC(═O)NHR¹⁰, O-tetrahydropyranyl, NR¹¹R¹², NR¹⁰C(═O)R⁹, NR¹⁰CO₂R⁹,NR¹⁰C(═O)NR¹¹R¹², NHC(═NH)NH₂, NR¹⁰S(O)₂R⁹, S(O)_(y)R⁹, CO₂R¹⁰,C(═O)NR¹¹R¹², C(═O)R⁹, CH₂OR¹⁰, CH═NNR¹¹R¹², CH═NOR¹⁰, CH═NR⁹,CH═NNHCH(N═NH)NH₂, S(═O)₂NR¹¹R¹², P(═O)(OR¹¹)₂, OR¹⁴, and amonosaccharide wherein each hydroxyl group of the monosaccharide isindependently either unsubstituted or is replaced by H, alkyl,alkylcarbonyloxy, or alkoxy;

X² is O, S, or NR¹⁰;

Y is selected from:

(a) a direct bond;

(b) optionally substituted CH₂, CH₂CH₂ or CH₂CH₂CH₂, wherein saidoptional substituents are one to about three R¹⁹ groups; and

(c) CH═CH, CH(OH)—CH(OH), O, S, S(═O), S(═O)₂, C(R¹⁸)₂, C═C(R¹⁹)₂,C(═O), C(═NOR²⁰) C(OR²⁰)R²⁰, C(═O)CH(R¹⁸), CH(R¹⁸)C(═O),C(═NOR²⁰)CH(R¹⁸), CHR²⁰ C(═NOR²⁰), C(═O)N(R²¹), N(R²¹)C(═O), CH₂Z, ZCH₂and CH₂ZCH₂, where Z is selected from C(R²⁰)₂, O, S, CO₂R²⁰, C(═NOR²⁰)and N(R²⁰);

R¹⁸ is independently selected from H, SO₂R^(18a), CO₂R¹⁸, C(═O)R^(18a)C(═O)NR¹⁸CR^(18d), optionally substituted alkyl, optionally substitutedalkenyl, and optionally substituted alkynyl;

R^(18a) is independently selected from optionally substituted alkyl,optionally substituted aryl, optionally substituted carbocyclyl andoptionally substituted heterocyclyl;

R^(18c) and R^(18d) are each independently selected from H andoptionally substituted alkyl, or together form a linking group of theformula (CH₂)₂—X¹—(CH₂)₂;

R¹⁹ is independently selected from R²⁰, thioalkyl, halogen, optionallysubstituted alkyl, optionally substituted alkenyl and optionallysubstituted alkynyl;

R²⁰ is independently selected from H, alkyl, OH, alkoxy, OC(═O)R^(18a) ,OC(═O)NR^(18c)R^(18d),OC(═S)NR^(18c)R^(18d), O(CH₂)_(p)NR^(18c)R^(18d),O(CH₂)_(p)OR²¹, optionally substituted arylalkyl, optionally substitutedheterocyclylalkyl and optionally substituted carbocyclyl;

R²¹ is independently selected from H and alkyl;

Q′ is selected from:

(a) a direct bond;

(b) NR⁶;

(c) optionally substituted CH₂, CH₂CH₂ or CH₂CH₂CH₂;

(d) CR²²R²⁴; and

(e) CH═CH, CH(OH)CH(OH), O, S, S(═O), S(═O)₂, C(═O), C(═NOR¹¹),C(OR¹¹)(R¹²), C(═O)CH(R¹³), CH(R¹³)C(═O), C(R¹⁰)₂, C(═NOR¹¹)CH(R¹³),CH(R¹³)C(═NOR¹¹), CH₂Z′, Z′—CH₂ and CH₂Z′CH₂;

Z′ is selected from C(R¹¹)(OR¹²), O, S, C(═O), C(═NOR¹¹) and NR¹¹;

R⁶ is selected from H, SO₂R^(2a), CO₂R^(2a), C(═O)R^(2a),C(═O)NR^(1c)R^(1d), optionally substituted alkyl, optionally substitutedalkenyl, and optionally substituted alkynyl, wherein said optionalsubstituents are one to about three R⁵ groups; or

alternatively, when Q is NR² and Q′ is NR⁶ or C(R¹⁰)₂, R² and R⁶ or oneof R¹⁰ are joined together to form:

wherein R⁷ and R⁸ are each independently selected from H, OH, alkyl,alkoxy, optionally substituted arylalkyl, optionally substitutedheteroarylalkyl, (CH₂)_(p)OR¹⁰, (CH₂)_(p)OC(═O)NR¹¹R¹² and(CH₂)_(p)NR¹¹R¹²; or R⁷ and R⁸ together form a linking group of theformula CH₂—X³—CH₂; X³ is a bond, O, S, or NR¹⁰;

J is selected from a bond, O, CH═CH, S, C(═O), CH(OR¹⁰), N(R¹⁰),N(OR¹⁰), CH(NR¹¹R¹²), C(═O)N(R¹⁷), N(R¹⁷)C(═O), N(S(O)_(y)R⁹),N(S(O)_(y)NR¹¹R¹²), N(C(═O)R¹⁷), C(R¹⁵R¹⁶), N⁺(O⁻)(R¹⁰), CH(OH)CH(OH)and CH(O(C═O)R⁹)CH(OC(═O)R⁹);

J′ is selected from O, S, N(R¹⁰), N⁺(O⁻)(R¹⁰), N(OR¹⁰) and CH₂;

R¹³ is selected from alkyl, aryl and arylalkyl;

R¹⁴ is the residue of an amino acid after the hydroxyl group of thecarboxyl group is removed;

R¹⁵ and R¹⁶ are independently selected from H, OH, C(═O)R¹⁰, O(C═O)R⁹,alkyl-OH, alkoxy and CO₂R¹⁰;

R¹⁷ is selected from H, alkyl, aryl and heteroaryl;

R²² is

X⁵ and X⁶ are independently selected from O, N, S, CHR²⁶, C(OH)R²⁶,C(═O) and CH₂═C;

X⁷ and X⁸ are independently selected from a bond, O, N, S, CHR²⁶,C(OH)R²⁶, C(═O) and CH₂═C;

X⁹ and X¹⁰ are independently selected from a bond, O, N, S, C(═O) andCHR²⁶;

X¹¹ is a bond or alkylene optionally substituted with NR¹¹R¹² or OR³⁰;

R²³ is selected from H, OR²⁷, SR²⁷, R²² and R²⁸;

R²⁴ is selected from R, thioalkyl, and halogen;

R²⁵ is selected from R¹ and OC(═O)NR^(1c)R^(1d);

R²⁶ is selected from H, optionally substituted alkyl and optionallysubstituted alkoxy, wherein

(1) ring G contains 0 to about 3 ring heteroatoms;

(2) any two adjacent hydroxyl groups of ring G can be joined to form adioxolane ring;

(3) any two adjacent ring carbon atoms of ring G can be joined to form afused aryl or heteroaryl ring; with the provisos that:

(a) when X¹¹ is a bond, ring G can be heteroaryl; and

(b) ring G:

(i) contains at least one carbon atom that is saturated;

(ii) does not contain two adjacent ring O atoms;

(iii) contains a maximum of two C(═O) groups;

R²⁷ is selected from H and alkyl;

R²⁸ is selected from aryl, arylalkyl, SO₂R²⁹, CO₂R²⁹, C(═O)R²⁹,optionally substituted alkyl, optionally substituted alkenyl andoptionally substituted alkynyl;

R²⁹ is selected from alkyl, aryl and heteroaryl;

R³⁰ is selected from H, alkyl, acyl and C(═O)NR¹¹R¹²;

m is independently selected from 0, 1, and 2;

p is independently selected from 1, 2, 3, and 4;

r is independently selected from 0, 1, and 2;

y is independently selected from 0, 1 and 2; and

z is selected from 0, 1, 2, 3 and 4;

with the provisos that at least one of Y and Q′ is a direct bond, when Yis a direct bond, Q′ is other than a direct bond, when Q′ is a directbond, Y is other than a direct bond, and when rings B and F are phenyl,G—X—W is CH₂NHC(═O), Y is a direct bond, Q is NR² and Q′ is NR⁶ where R⁶is joined with R² to form

then R³ is other than CH₂SCH₂CH₃.

In an alternative embodiment, the methods of the present invention mayinvolve a compound of Formula II which is a small organic molecule thatis a derivative of the indolocarbazole K-252a. The compound of FormulaII has been shown to prevent death of motorneurons in vitro byinhibiting the JNK signaling pathway associated with stress and injury(Maroney et al., J. Neuroscience, 18(1), 104-111, 1998).

The compound of Formula II is a bis-thioethylmethyl analog of K-252a.Modifications of Formula II which retain functional activity are alsocontemplated. As used herein, the term “functional activity” refers tothe ability of the composition to prevent hearing loss, prevent loss ofsense of balance, prevent the death of sensory hair cells, preventsudden sensorineural hearing loss due to the loss of sensory hair cells,preserve function of sensory hair cells prior to or subsequent totrauma, treat damaged sensory hair cells, prevent death of cochlearneurons. The compound of Formula II has an indolocarbazole skeleton andthe structure shown below.

The present invention also provides methods for preventing hearing lossin a subject comprising administering to said subject an effectiveamount of the compound of Formula II;

The present invention further provides methods for preventing loss ofsense of balance in a subject comprising administering to said subjectan effective amount of the compound of Formula II;

FORMULA II

The present invention further provides methods for preventing death ofsensory hair cells in a subject comprising administering to said subjectan effective amount of the compound of Formula II;

The present invention further provides methods for preventing suddensensorineural hearing loss in a subject due to death of sensory haircells comprising administering to said subject an effective amount ofthe compound of Formula II, defined above.

The present invention also provides methods for preserving function ofsensory hair cells prior to or subsequent to trauma in a subjectcomprising administering to said subject an effective amount of thecompound of Formula II, defined above.

The present invention further provides methods for preventing death ofcochlear neurons in a subject comprising administering to said subject atherapeutically effective amount of the compound of Formula II, definedabove.

Indolocarbazoles are generally lypophilic and, therefore, are able tocross biological membranes much more readily than proteins. Further,indolocarbazoles can have much longer half lives in vivo than dopolypeptides.

K-252a derivatives have promise for disorders such as Alzheimer'sdisease, motor neuron disease, Parkinson's disease, Huntington's diseaseand epilepsy (See, for example, U.S. Pat. No. 5,461,146, issued Oct. 24,1995; U.S. Pat. No. 5,621,100, issued Apr. 15, 1997).

The compounds of Formula I and Formula II may also be useful in thetreatment of peripheral or central nerve disorders, and cytokineoverproduction (See PCT/WO97/49406, International Publication Date Dec.31, 1997, the disclosures of which are hereby incorporated herein byreference, in their entirety).

In another alternate embodiment, the methods of the present inventionmay involve compounds which are intermediates in the preparation of thecompounds of Formulas I and II. Examplary of such intermediate compoundsinclude, for example, the compounds identified as 5 in Example 11, and1, 2, 3, 4, 5, 6, 7, and 8 in Example 12. A particularly preferredintermediate compound is compound 7 in Example 12.

The cochlea is an useful biological model system for studies on theprotective therapeutic potential of various substances on sensory haircells. The function of the cochlea can be accurately monitored at twolevels; at the level of the organ of Corti by measuring cochlearmicrophonic potentials and otoacoustic emissions, and at the level ofthe cochlear nerve by measuring compound action potentials, summatingpotentials, or auditory brainstem responses, ABRs. Additionally, thequantity of sensory hair cells and cochlear neurons in the Organ ofCorti is well known and trauma-induced changes can be accuratelyevaluated by morphometric methods including cytocochleograms andneuronal counts. Following experimentally-induced lesions, the patternof degeneration and the sequence of events in the mammalian auditoryorgan are well known. The sensory hair cells are the primary targets ofnoise and ototoxic drugs. We know today what kind of noise or dosage ofototoxic drugs induces destruction of sensory hair cells at locations inthe inner ear. When the sensory hair cells are destroyed, theinnervating cochlear neurons degenerate secondarily.

The fused pyrrolocarbazoles of the present invention may exist inprodrug form. As used herein, the term “prodrug” is intended to includeany covalently bonded carriers which release the active parent drugaccording to Formulas I and/or II or other formulas or compounds of thepresent invention in vivo when such prodrug is administered to amammalian subject. Since prodrugs are known to enhance numerousdesirable qualities of pharmaceuticals (e.g., solubility,bioavailability, manufacturing, etc.), the compounds of the presentinvention may be delivered in prodrug form. Thus, the present inventioncontemplates prodrugs of the claimed compounds, compositions containingthe same, and methods of delivering the same. Prodrugs of a compound ofthe present invention, for example Formulas I and II, may be prepared bymodifying functional groups present in the compound in such a way thatthe modifications are cleaved, either in routine manipulation or invivo, to the parent compound.

Accordingly, prodrugs include, for example, compounds of the presentinvention wherein a hydroxy, amino, or carboxy group is bonded to anygroup that, when the prodrug is administered to a mammalian subject,cleaves to form a free hydroxyl, free amino, or carboxylic acid,respectively. Examples include, but are not limited to, acetate, formateand benzoate derivatives of alcohol and amine functional groups; andalkyl, carbocyclic, aryl, and alkylaryl esters such as methyl, ethyl,propyl, iso-propyl, butyl, isobutyl, sec-butyl, tert-butyl, cyclopropyl,phenyl, benzyl, and phenethyl esters, and the like.

The pharmaceutically acceptable salts of the present invention can besynthesized from the parent compound which contains a basic or acidicmoiety by conventional chemical methods. Generally, such salts can beprepared by reacting the free acid or base forms of these compounds witha stoichiometric amount of the appropriate base or acid in water or inan organic solvent, or in a mixture of the two. Generally, nonaqueousmedia like ether, ethyl acetate, ethanol, isopropanol, or acetonitrileare preferred. Lists of suitable salts are found in Remington'sPharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa.,1985, p. 1418, the disclosure of which is hereby incorporated byreference.

The compounds of the present invention may be prepared in a number ofways well known to those skilled in the art. The compounds can besynthesized, for example, by the methods described below, or variationsthereon as appreciated by the skilled artisan. All processes disclosedin association with the present invention are contemplated to bepracticed on any scale, including milligram, gram, multigram, kilogram,multikilogram or commercial industrial scale.

It will be appreciated that the compounds of the present invention maycontain one or more asymmetrically substituted carbon atoms, and may beisolated in optically active or racemic forms. Thus, all chiral,diastereomeric, racemic forms and all geometric isomeric forms of astructure are intended, unless the specific stereochemistry or isomericform is specifically indicated. It is well known in the art how toprepare and isolate such optically active forms. For example, mixturesof stereoisomers may be separated by standard techniques including, butnot limited to, resolution of racemic forms, normal, reverse-phase, andchiral chromatography, preferential salt formation, recrystallization,and the like, or by chiral synthesis either from chiral startingmaterials or by deliberate synthesis of target chiral centers.

As will be readily understood, functional groups present may containprotecting groups during the course of synthesis. For example, aminoacid side chain substituents of the present compounds may be substitutedwith protecting groups such as benzyloxycarbonyl or t-butoxycarbonylgroups. Protecting groups are known per se as chemical functional groupsthat can be selectively appended to and removed from functionalities,such as hydroxyl groups and carboxyl groups. These groups are present ina chemical compound to render such functionality inert to chemicalreaction conditions to which the compound is exposed. Any of a varietyof protecting groups may be employed with the present invention.Preferred protecting groups include the benzyloxycarbonyl (Cbz; Z) groupand the tert-butyloxycarbonyl (Boc) group. Other preferred protectinggroups according to the invention may be found in Greene, T. W. andWuts, P. G. M., Protective Groups in Organic Synthesis 2d. Ed., Wiley &Sons, 1991.

Fused pyrrolocarbazoles, such as indolocarbazoles may be synthesized bymethods taught, for example, in U.S. Pat. Nos. 4,923,986; 4,877,776;5,093,330; 5,461,146; 5,468,872; 5,621,100; 5,621,101; 5,516,771; and5,599,808; and PCT publication Nos. WO 93/08809 and WO 97/46565, thediscloses of which are hereby incorporated herein by reference, in theirentireties. Additional methods of preparation are set forth in Moody etal., J. Org. Chem. 57:2105-2114 (1992), also incorporated herein byreference.

Fused pyrrolocarbazoles, such as indenocarbazoles, as well as additionalcompounds wherein Q′ is not a single nitrogen, may be synthesized bymethods taught, for example, in U.S. Pat. Nos. 5,475110; 5,591,855;5,594,009; 5,705,511; 5,616,724; and 5,801,190; the disclosures of whichare hereby incorporated herein by reference in their entireties.

Fused pyrrolocarbazoles, such as bridged indenocarbazoles, may beprepared by methods taught, for example, in copending U.S. patentapplication Ser. No. 09/325,140, the disclosure of which is herebyincorporated herein by reference in its entirety.

Fused pyrrolocarbazoles, such as cyclic substituted pyrrolocarbazolesand isoindolones, may be prepared by methods taught, for example, incopending U.S. Provisional Application Ser. No. 60/119,834, thedisclosures of which are hereby incorporated by reference, in theirentireties.

The invention is set forth in more detail in the examples below. Thefollowing examples are meant to illustrate the invention and are not tobe construed to limit the invention in any way. Those skilled in the artwill recognize modifications that are within the spirit and scope of theinvention. All references cited herein are hereby incorporated byreference in their entirety.

EXAMPLE 1 Prevention of Hearing Impairment/Cell Death

Baseline hearing of 8 pigmented guinea pigs (200-300 g) was tested byABR as described below. ABR is a measure of hearing function. Fouranimals were treated with daily subcutaneous (s.c.) injections of thecompound of Formula II, 1 mg/kg, starting 24 hours prior to noise traumaand lasting until 21 days after exposure to the noise trauma. Fouranimals served as controls and received only vehicle (the fourth animaldied under anaesthesia). All the animals were exposed to traumaticoctave band noise, center frequency 4.0 kHz, 125 dB SPL (sound pressurelevel) for 105 min.

ABR thresholds were again determined 21 days after exposure to the noisetrauma. ABR thresholds were measured with System II hardware and BioSigsoftware (Tucker Davis Technology). Stimulus with 1 ms cos2 rise andfall and 18 ms plateau was presented with Beyer earphone connected tospeculum (placed in the meatus of the external ear canal) at a rate of20 Hz. Stimulus was calibrated against a Bruel & Kjaer 4134 microphoneconnected to a Bruel & Kjaer 2203 sound level meter. A Grass P15preamplifier and custom amplifier provided gain of 10⁵ and filtering of0.3 to 3.0 kHz. Two responses were obtained at each intensity level.Thresholds were determined for the frequencies 2, 4, 8, 16 and 32 kHzfrom a set of responses at varying intensities with 5 dB intervals and1000 sweeps near threshold.

After the treatment period of 21 days and measurement of ABRs, theanimals were sacrificed and the temporal bones fixed by 4% PFA(paraformaldehyde) through perilymphatic perfusion. The left cochleaswere dissected for surface specimens and the number of sensory haircells in each auditory organ was evaluated by cytocochleograms. Theupper halves of the cochleas were processed for cytocochleograms withoutembedding by staining cochlear whole mounts with rhodamine phalloidine(Molecular Probes, Eugene, Ore.) in PBS containing 0.25% Triton X-100overnight at 4° C and mounted in Vectashield (Vector). Phalloidin is aspecific marker for cellular F-actin. Basal halves of the cochleas wereembedded in

Epon and processed for cytocochleograms. Selected sections ofEpon-embedded cochlear turns were processed for fine structural study byelectron microscopy.

Results

Noise exposure caused a hearing loss in all guinea pigs (four in eachgroup) as measured by ABR (FIG. 1). At all frequencies except 32 kHz,the treated animals had an intermediate shift in their ABR measurement.The threshold shift was 40-60 dB SPL across the test frequencies incontrol (vehicle-treated, noise exposed) animals but only 20-40 dB inanimals receiving the compound of Formula II.

Before noise lesioning, the total number of OHCs (or Deiters scars, thesites where OHCs had been located) was equivalent in control andtreatment groups. The total number of OHCs ranged from about 6000 to7000, averaging 6583+/−203 sensory hair cells in the control group and6445+/−234 sensory hair cells in the treated group. In allnoise-lesioned, control cochleas, the organ of Corti showed extensivedegeneration of outer sensory hair cells throughout the cochlea, with17-84% of the cells lost (Table 2). The most affected area was between11 mm and 7 mm from the Round Window (RW) located in the middle turn andupper basal turn. Within this region, hair cells were absent over adistance of about 1.5-3.0 mm (FIG. 2b).

In contrast, three out of four compound of Formula II treated animalsshowed only a minor loss of sensory hair cells ranging from 1-3% (Table2). The fourth animal had a loss of 19%. In the three animals withalmost complete sensory hair cell preservation, the small percentage ofcells lost did not appear to localize to a particular site in the organof Corti (FIG. 2a). The light microscope morphology of the preservedsensory hair cells in the treated animals appeared normal and showedonly minor changes by electron microscopy.

In this noise-trauma model, both the functional measure of the ABR andthe actual cell counting suggest that the compound of Formula II waseffective in preserving both the outer sensory hair cells themselves andtheir functional abilities.

TABLE 2 LOST OUTER SENSORY CELLS (OHC) Total TOTAL % OHC TreatmentAnimal ID# OHC OCH1 OCH2 OCH3 OHC LOST LOST Formula II 13 6036 90 25 71186 3.1% Formula II 14 6477 19 3 27 49 0.8% Formula II 19 6990 36 18 46100 1.4% Formula II 20 6276 356 381 428 1195 19.0% MEAN 6445 133 107 143383 6.1% SD 234 99 106 110 314 5.0% CONTROL 15 6855 635 712 760 210730.7% CONTROL 17 6282 1554 1832 1863 5249 83.6% CONTROL 18 6612 351 341411 1103 16.7% MEAN 6583 547 962 1011 2820 43.7% SD 203 445 549 536 152924.9%

EXAMPLE 2 Prevention of Hearing Impairment/Apoptosis

Noise lesioning.

Adult Dunkin-Hartley female guinea pigs (weight 300-500 g) were exposed,4 animals at a time, to octave band noise, with a center frequency of4.0 kHz, 120 dB SPL for 6 hr as described in Example 1. Fragmented haircell nuclei were assessed from noise-exposed cochleas immediately and 1,2, 4, 6, 8 and 14 days after noise exposure. Noise-exposed andnonexposed guinea pigs were decapitated under deep anesthesia and innerears were perilymphatically perfused with 4% paraformaldehyde in PBS,and processed for 5-mm-thick paraffin-embedded sections (Ylikoski etal., Hear Res 65:69-78, 1993; Ylikoski et al., Hear Res 124:17-26,1998). TUNEL-staining was performed as described below. DNAfragmentation was also verified by DAPI nuclear counterstain (seebelow). Further, trauma-induced nuclear fragmentation was verified bymorphological analysis of contralateral cochleas that wereperilymphatically fixed with 2.5% glutaraldehyde in 0.1M phosphatebuffer, pH 7.4, postfixed with 1% osmium tetroxide, embedded in epoxyresin, and prepared for surface preparations (see below). Selectedcochlear segments were then cut in transverse and horizontal planes tosemi-thin (1.0 mm) sections that were stained with 1% toluidine blue.Analysis was done under an Olympus Provis microscope equipped withNomarski optics.

Test-substance delivery.

Formula II was injected subcutaneously to noise-exposed guinea pigs atthe dose of 1 mg/kg, starting 2 hr before noise exposure and continuingdaily for 2 weeks. Formula II was dissolved in 5% Solutol (BASF Corp.,Parsippany, N.J.) in PBS, pH 7.4. The 1 mg/kg dose of the compound ofFormula II was prepared daily from a 10 mg/ml stock in 25% Solutol thatwas stored at 4° C. Noise-exposed control guinea pigs received thevehicle alone.

Hearing tests.

Auditory brainstem responses (ABRs) were measured 2 days before noiseexposure, and 2, 6 and 14 days after exposure under anesthesia (xylazine10 mg/kg and ketamine 40 mg/kg). One ms hanning windowed binauralstimuli with 0.5 ms rise and fall of frequencies 2, 4, 6, 8, 16 and 32kHz were presented at a rate of 10 Hz. Preamplifier and custom dataacquisition provided a gain of 80000, with filtering of 0.3 to 10 kHz.Two responses summed from 1000 trials were obtained at each intensitylevel. Hearing threshold was determined from a set of responses atvarying intensities. The threshold was defined as the last appearance ofpeak 3 or 4 in the ABR waveforms. Testing was performed in a soundattenuated box and animal temperature was kept at 37° C.

Cytocochleograms.

Two weeks after noise exposure, control and Formula II-treated guineapigs were decapitated under deep anesthesia and inner ears wereperilymphatically perfused with 2.5% glutaraldehyde in 0.1M phosphatebuffer, pH 7.4, postfixed with 1% osmium tetroxide, embedded in epoxyresin and processed for surface preparations as earlier described indetail (Ylikoski, Acta Otolaryngol (Stockh) Suppl 326:5-20, 1974).Briefly, segments of the cochlear duct were dissected free from themodiolus, trimmed and re-embedded in resin. The re-embedded cochlearsegments were studied as surface preparations under an Olympus Provismicroscope equipped with Nomarski optics. Hair cells were characterizedas missing if both no cuticular plate and stereocilia in the appropriatelocation was observed. For cell counting, a 10×10 square eye reticularand a 40×objective lens were used. The percentage of missing hair cellswas plotted as a function of the percentage length of the organ ofCorti.

Results

Hair cells die by apoptosis after noise trauma

Noise trauma induced maximal damage to hair cells of the organ of Cortiat the 9 mm region from the round window (second cochlear turn.Noise-exposed (n=16) and nonexposed (n=3) cochleas were cut intransverse (midmodiolar) plane, and double stained with the DAPIcounterstain (FIG. 3a) and with TUNEL-method (FIGS. 3b,c). In addition,resin-embedded organ of Corti's of noise-exposed cochleas (n=6) were cutin transverse (FIG. 3d) or horizontal plane (FIGS. 3e,f), and stainedwith toluidine blue. Most of the TUNEL-labeled cells were located in thearea of maximal lesion, the second cochlear turn (FIG. 3e). During thestudy period, the first 2 weeks after noise exposure, TUNEL-labeling wasnot detected in the supporting cells of the organ of Corti (FIGS. 3a,b)or in the cochlear neurons. In non-exposed cochleas, cells of the organof Corti as well as neurons were not TUNEL-labeled. Two weeks afternoise exposure, no TUNEL-labeled sensory hair cells could be found incontrol or treated cochleas.

In control cochleas, the main part of hair cell apoptosis occurredduring the first 6 days after noise exposure. The apoptotic profilesconcentrated to the upper basal and lower middle coil, in accordancewith the results of cytocochleograms showing that sensory hair cell losswas concentrated to this area (about 50% distance from the apex). Theamount of apoptotic hair cell loss was less in Formula II-treatedcochlea, these results being in agreement with the ABR-measurementsshowing a statistically significant difference in hearing functionbetween the two group at this postexposure time point.

Formula II attenuates noise-induced hearing loss in vivo

Hearing thresholds measured 2 days before noise exposure (baselinevalues) showed no significant difference between the noise-exposedcontrol and treated group. Noise exposure caused threshold shifts in allguinea pigs as seen 2 days after the exposure (noise-exposed controln=4, Formula II-treated n=5) (FIG. 4a). By day 6 postexposure, thresholdshifts were significantly less in the Formula II group (n=11) than inthe noise-exposed control group (n=8, P<0.05 at 4 and 8 kHz, P<0.01 at16 kHz) (FIG. 4b). By 2 weeks postexposure, the difference between the 2groups became even more pronounced (noise-exposed control n=17, FormulaII-treated n=17) (FIG. 4c). At this stage, ABR threshold shifts rangedfrom 28 to 45 dB SPL in noise-exposed controls, and from 12 to 22 dB SPLin Formula II-treated animals (P<0.01 at all frequencies).

Formula II attenuates noise-induced hair cell loss

Two weeks after noise exposure and after final ABR recordings had beenperformed, the numbers of preserved and missing hair cells were assessedin resin-embedded cochlear surface preparations. Total number of OHCsper guinea pig cochlea (lost plus preserved hair cells) ranged from 6240to 6897 (Table 3). There was a large variation in the amount of haircell damage in noise-exposed control cochleas in which the extent oflost hair cells ranged from 305 to 1266 (n=13). In Formula II treatedcochleas, the number of missing hair cells ranged from 106 to 632 (n=13)(Table 3). This difference is statistically significant (P<0.01).

TABLE 3 Sample OHCs Lost hair cells ORC loss I.D. total IHC OHC1 OHC2ORC3 total (%) Formula 6897 1 40 27 38 105 (1.5) II-1 Formula 6672 7 7262 63 197 (3.0) II-2 Formula 6699 24 20 38 47 105 (1.6) II-3 Formula6573 28 42 67 74 183 (2.8) II-4 Formula 6585 19 53 37 64 154 (2.3) II-5Formula 6666 17 41 50 54 145 (2.2) II-6 Formula 6846 14 61 53 76 190(2.8) II-7 Formula 6723 17 64 66 80 210 (3.1) II-8 Formula 6795 24 57 6093 210 (3.1) II-9 Formula 6748 9 38 45 58 141 (2.1) II-10 Formula 6387 674 74 56 204 (3.2) II-11 Formula 6240 2 59 69 161 289 (4.7) II-12Formula 6454 25 180 198 229 607 (9.4) II-13 Control- 6621 7 96 156 255507 (7.7) 1 Control- 6421 33 77 109 178 364 (5.7) 2 Control- 6480 77 89136 141 366 (5.6) 3 Control- 6690 56 118 119 225 462 (6.9) 4 Control-6687 23 95 92 95 282 (4.2) 5 Control- 6630 43 125 111 139 375 (5.7) 6Control- 6897 56 102 139 184 425 (6.2) 7 Control- 6882 78 156 222 341 719 (10.4) 8 Control- 6507 77 205 117 184 506 (7.8) 9 Control- 6690 117222 413 514 1149 (17.2) 10 Control- 6621 7 96 156 255 507 (7.7) 11Control- 6423 2 65 149 114 328 (5.1) 12 Control- 6849 11 161 209 281 648(7.6) 13

In vehicle-treated, noise-exposed control cochleas, in addition tomissing hair cells, there were several distorted OHCs with irregularconfiguration and disarrayed stereocilia, but a preserved cuticularplate and nucleus in regions adjacent to the site of maximal damage.Most of the noise-exposed control cochleas showed a relatively welldemarcated area of maximal damage, extending over 0.5 to 1.0 mm in theregion of about 9 mm from the round window. In addition, scattered OHCloss was found particularly along the entire upper half of the cochlea.Formula II treated cochleas showed some destruction of the organ ofCorti, but usually in an area extending over 0.1-0.2 mm only. Alsoscattered OHC loss was less than in noise-exposed control cochleas. Thecytocochleograms (FIGS. 5a,b) illustrate the average percentages ofmissing IHCs and OHCs along the length of the organ of Corti innoise-exposed control (n=13) and Formula II treated (n=13) cochleas.These results indicate that, in our intense noise paradigm, Formula IIrescues IHCs as well as OHCs of each of the 3 rows.

EXAMPLE 3 Protective Effect of the Compound of Formula II AgainstNeomycin in Neonatal Rat Cochlear Explants

The compound of Formula II attenuated neomycin-induced cochlear sensoryhair cell loss in vitro. The effect of the compound of Formula II onneomycin-induced hair cell degeneration was determined in organotypiccultures of the neonatal organ of Corti.

Cochlear cultures.

The basal half of cochleas containing the basal and middle turns weredissected from postnatal day 2 Wistar rats. The cultures were maintainedon Nuclepore filters (pore size 0.1 mm; Pleasanton, Calif.) placed on ametal grid in F12 medium (Life Technologies, Gaithersburg, Md.)containing 15% fetal bovine serum (Life Technologies). After a2-hour-long stabilization period, explants were exposed to 100 mMneomycin sulfate (Sigma, St Louis, Mo.) for 48 hr. Formula II (500 nM)was added at the time of initiation of the cultures and every 12 hrthereafter.

Hair cell counts in cochlear cultures.

Explants were fixed with 4% paraformaldehyde/0.5% glutaraldehyde inphosphate buffered saline (PBS), pH 7.4, for 30 min and dissected forsurface preparations. They were stained with a 1:100 dilution ofrhodamine-phalloidin in PBS containing 0.25% Triton X-l 00 overnight at4° C. and mounted in Vectashield (Vector). Outer hair cell (OHC) numberswere quantified with a Zeiss Axiovert 100/135 epifluorescent microscope(Germany) connected to a Bio-Rad MRC-1024 confocal laser scanning system(Richmond, Calif.). Hair cells were characterized as missing if nostereocilia and cuticular plate were observed. Numbers of OHCs wereevaluated using a 40×objective lens and an ocular grid. Nine to 1 fieldsfilled by 30 OHCs in each of the 3 rows (when all of them were present)were studied from each explant. Basal and middle coils were analyzedseparately. Three separate experiments, each including 4 explants ofboth conditions (neomycin and neomycin plus Formula II) were analyzed.

Results

When the cultures were treated with 100 μM neomycin, many sensory haircells were lost within 48 hours, especially in the basal half of thecochlea. 100 μM neomycin caused the loss of most basally located sensoryhair cells, as detected in surface preparations of the cultures usingF-actin (phalloidin) as a sensory hair cell marker (FIG. 6a). Theseresults are in accordance with earlier data showing that sensory haircells situated in the basal part of the cochlea are more sensitive toantibiotics than the apically located ones. When 500 nM of the compoundof Formula II was added to the cultures together with neomycin, sensoryhair cell degeneration was reduced (FIG. 6b).

In explants exposed to 100 mM neomycin for 48 hr (n=12), severe haircell degeneration occurred in the basal turn of the cochlea (FIG. 7a),as shown in phalloidin-labeled confocal images. Formula II preventedhair cell loss in the basal turn of the cochlea (FIG. 7b). Approximately90% of OHCs in the basal turn and 25% of OHCs in the middle turn werelost (FIG. 7c). In cultures co-incubated with 500 nM Formula II andneomycin for 48 hr (n=12), about 90% of OHCs in the basal turn werepreserved.

EXAMPLE 4 Protective Effect of the Compound of Formula II AgainstGentamicin Toxicity in Cochlear and Vestibular Hair Cells

Animals, tissues, lesioning and test drug delivery.

Adult Dunkin-Hartley female guinea pigs (weight 300-400 g) were used.They were given free access to water and a regular guinea pig diet. Theanimals were given 1 week of adjustment before baseline ABR recordingsand gentamicin (GM) treatment.

Two experimental groups were formed. Group 1 guinea pigs served ascontrol and were treated with gentamicin only. The animals were injecteds.c. with gentamicin at 120 mg/kg body weight, once daily for 14 days.Three guinea pigs (yielding a total of 6 inner ears) were included inthis group. The animals were decapitated 30 days after the start of theinjections.

Group 2 guinea pigs were treated with gentamicin plus Formula II. Theanimals were injected s.c. with GM at 120 mg/kg body weight, plusFormula II at 1 mg/kg, once daily for 14 days. Formula II treatmentstarted 1 day before GM injections. Formula II treatment was continuedfor 28 or 29 days. Four guinea pigs (8 inner ears) were included in thisgroup. The animals were decapitated 30 days after the start of GMinjections.

In addition to the animals of groups 1 and 2, two guinea pigs thatreceived only GM and two guinea pigs that received GM plus Formula IIwere decapitated at day 14. These inner ears were used for thedocumentation of apoptosis. A large number of apoptotic vestibular andcochlear hair cells were found in only GM-treated inner ears(TUNEL-staining) .

Evaluation of auditory function.

ABRs were determined 1 day before GM injections (baseline values) and atday 30 after the start of the GM injections. Threshold shift shows thedifference between the baseline and the final threshold. Thresholds weredetermined at frequencies 2, 4, 8, 16 and 32 kHz from a set of responsesat varying intensities with 5 dB intervals and 1000 sweeps near thethreshold. ABRs were measured with System II hardware and BioSigsoftware. Ketamine (40 mg/kg) and xylazine (10 mg/kg) were used foranaesthesia.

Processing of the inner ears for morphometric analyses.

Guinea pigs were decapitated under deep anaesthesia and the inner earswere perilymphatically fixed and immersed with 2.5% glutaraldehyde in0.1 M phosphate buffer (pH 7.4), postfixed with 1% osmium tetroxide, andembedded in Epon. Cochleas were processed for cytocochleograms(Ylikoski, Acta Otolaryngol (Stockh) Suppl 326:5-20, 1975). Theseparately dissected and Epon-embedded vestibular organs were cut for 1μm-thick sections. Every 25th section was collected onto slides andstained with 1% toluidine blue. The quantity of hair cell loss investibular organs was evaluated by counting hair cells in at least 10corresponding transverse sections of the horizontal or superiorampullary cristae. Two control (GM-treated) and 3 Formula II plusGM-treated ampullae were used for analysis. The presence of cell nucleiin the sensory layer (upper half) of the sensory epithelium was regardedas the presence of hair cells. Supporting cell nuclei were clearlydistinguished from hair cells by their position (lower part) in theepithelium and by toluidine blue that stains the hair cells weaker thanthe supporting cells. Hair cell loss was measured by comparing thenumber of hair cell nuclei in the sensory layer to the number of haircell nuclei in the basal cell layer.

Results

Formula II attenuates gentamicin-induced hearing loss.

Compared to control cochleas (GM only, n=6), Formula II treated cochleas(n=8) showed less threshold shift at all frequencies tested. Thedifference was statistically significant at all frequencies tested(P<0.01). The average threshold shifts are shown in FIG. 8.

Formula II attenuates hair cell loss in the vestibular end organs aftergentamicin intoxication.

Compared to control (GM only) ampullae (n=2), Formula II treatedampullae (n=3) showed less hair cell loss, as revealed by the analysisof relative hair cell loss (Table 4). The protective effect of FormulaII in the ampullae was 70%.

TABLE 4 Relative numbers of preserved hair cells in GM and GM + FormulaII treated cochleas Relative length of Number of sensory epitheliumTotal number Hair cells Sample sections (frames) of hair cells per frameControl 11 169 199 1,2 Control 16 245 270 1,1 Formula II 17 276 518 1,9Formula II 10 174 328 1,9 Formula II 23 287 620 2,2

EXAMPLE 5 Protective Effect of the Compound of Formula II Against Deathof Cochlear Neurons in Vitro

Dissociated neuronal cultures.

Neuronal enriched cultures from embryonic day 21 (E21) rat cochlearganglia were prepared as previously described (Ylikoski et al., Hear.Res., 1998, 124:17-26). The cultures were maintained in F14 medium (LifeTechnologies, Gaithesburg, Md.) containing 10% horse serum (LifeTechnologies). Formula II (500 nM), neurotrophin-3 (NT-3; 2 ng/ml,Promega, Madison, Wisc.) or nerve growth factor (NGF; 2 ng/ml, Promega)was added at the beginning and after each 12 hr to the medium. After 48hr, cultures were fixed with 4% paraformaldehyde and responses wereassessed under a phase contrast microscope. Surviving neurons weredistinguished by a phase-bright cell body and definite neurites.

Results

Formula II attenuated the death of cochlear neurons in vitro. NT-3 isthe most potent neurotrophic factor promoting survival of dissociatedcochlear neurons of the perinatal rat, and NGF does not have any effector its effect is very weak (Ylikoski et al., 1998). As assessed inparallel cultures, Formula II was as efficacious as NT-3 in promotingsurvival of cochlear neurons. NGF served as a negative control (FIG. 9).

EXAMPLE 6 Delayed Administration of Formula II After Noise Trauma

Guinea pigs were exposed to 120 dB, 4 kHz noise for 6 hr. The compoundof Formula II was administered beginning 2 hours to 1 day prior to noisetrauma, or 1 day after noise trauma, or 4 days after noise trauma.Dosing continued for 2 weeks after noise exposure. As described above,cochleas were prepared and numbers of preserved and lost inner haircells (IHCs) and outer hair cells of different rows (OHC1, OHC2, OHC3)were counted 2 weeks post noise trauma.

Evaluation of auditory function.

ABRs were determined 1 day before noise trauma (baseline values) and 2weeks post noise trauma. Threshold shift shows the difference betweenthe baseline and the final threshold. Thresholds were determined atfrequencies 2, 4, 8, 16 and 32 kHz from a set of responses at varyingintensities with 5 dB intervals and 1000 sweeps near the threshold. ABRswere measured with System II hardware and BioSig software. Ketamine (40mg/kg) and xylazine (10 mg/kg) were used for anaesthesia.

Results

Formula II-treated animals showed less threshold shift at allfrequencies tested than did vehicle-treated, noise-exposed controls. Theaverage threshold shift of control animals is 20-40 dB greater than thatof animals treated with Formula II beginning prior to the lesion. Thesedata are presented in FIG. 10 and also, separately, as FIG. 4c.Decreased threshold shifts are also noted if administration of CEP-1347is begun 1 or 4 days after exposure to noise. These data are presentedin FIG. 10.

Compared to control cochleas, Formula II-treated cochleas showed lessloss of hair cells following exposure to noise. The effect of delayeddosing of Formula II on hair cell loss is shown in FIG. 11; the top andbottom graphs were presented separately as FIG. 5. A statisticaldifference existed in hair cell loss between the 1 day delayed group(n=6) and noise-treated control group (n=13) P<0.01. A statisticaldifference existed between the 4 day delayed group (n=1 1) andnoise-treated control group (n=13); P<0.05. Results are set forth inTable 5.

TABLE 5 OHCs Lost hair cells OHC loss Sample total IHC OHC1 OHC2 OHC3total (%) 1 day 6900 0 35 46 58 139 (2.0) delayed 1 day 6528 54 129 116106 351 (5.4) delayed 1 day 6708 1 44 33 8247 159 (2.4) delayed 1 day6744 32 115 117 200 432 (6.4) delayed 1 day 6864 46 82 105 67 254 (3.7)delayed 1 day 6864 0 30 13 42  85 (1.2) delayed 4 days 6843 34 259 300327  886 (12.9) delayed 4 days 6312 56 177 247 205  629 (10.0) delayed 4days 6606 0 30 22 60 112 (1.8) delayed 4 days 6732 54 173 167 272 612(9.1) delayed 4 days 6531 36 73 105 141 319 (4.9) delayed 4 days 6792 372 15 25 112 (1.6) delayed 4 days 6225 2 62 45 36 143 (2.3) delayed 4days 6936 44 124 111 117 352 (5.2) delayed 4 days 6777 7 287 172 283 742 (10.9) delayed 4 days 6555 54 175 170 245 590 (9.0) delayed 4 days6441 10 120 97 225 442 (6.9) delayed

Compared to control cochleas, Formula 11-treated cochleas showed lessthreshold shift at all frequencies tested than did control. The averagethreshold shifts are shown in FIG. 10. The most significant differencefrom control was exhibited by the group of animals which wereadministered Formula II before noise trauma. Both 1 day and 4 daydelayed adminsitration of Formula exhibited less threshold shift thandid the control group. Compared to control cochleas, Formula II-treatedcochleas showed less hair cell loss. The effect of delayed dosing ofFormula II on hair cell loss is shown in FIG. 11.

EXAMPLE 7 Assessment of Hair Cell Death and Immunohistochemistry inCochlear Cultures

Six, 12 and 24 hr after adding 100 μM neomycin to the medium, cochlearcultures were fixed with 4% paraformaldehyde in PBS for 30 min. Thespecimens were prepared for 5 um-thick paraffin sections. They werestained with a TUNEL-kit, mounted in Vectashield containing DAPI nuclearcounterstain (Vector, Burlingame, Calif.), and viewed under an OlympusProvis microscope (Tokyo, Japan) using epifluorescence. In addition toTUNEL-staining, DNA fragmentation was verified under UV illuminationusing DAPI counterstain.

Adjacent sections were immunostained with a polyclonal phospho-c-Junantibody (Ser73, 1:500; New England Biolabs, Beverly, Mass.) and apolyclonal phospho-JNK antibody (Thr183/Tyr185, 1:250 dilution; NewEngland Biolabs). The phospho-JNK antibody detects the duallyphosphorylated isoforms of JNK 1, 2 and 3. The specificity of thephospho-specific antibodies was verified by Western blotting usingsorbitol-treated PC 12 cells. Phospho-JNK antibody recognized thephosphorylated p54/p46 JNK and phospho-c-Jun antibody the phosphorylatedp46 c-Jun in sorbitol-treated, but not in untreated PC 12 cells. Forimmunohistochemical detection, the avidin-biotin-peroxidase method(Elite ABC Kit; Vector) and diaminobenzidine were used. Stainings wereamplified using tyramide signal amplification (TSA-Indirect Kit, NENLife Science Products, Boston, Mass.) according to Brady et al. (1999),J. Neurosci., 19:2131-2142). No counterstaining was used in conjuctionwith the phospho-specific antibodies. In addition, a polyclonalcalbindin antibody (1:10000 dilution; Swant, Switzerland) was used as amarker for hair cells. Calbindin immunoreaction was detected using theABC method and diaminobenzidine without tyramide signal amplification,and the sections were lightly counterstained with 1% toluidine blue.Analysis was done under Olympus Provis microscope and bright-fieldoptics.

Results

Formula II is effective in blocking JNK activation in stressed haircells

Hair cell death and involvement of the JNK signaling cascade inorganotypic cochlear explants of neonatal rats was examined. In paraffinsections of normal (nontreated) explants, the normal cellulararchitecture of the organ of Corti, one row of IHCs and 3 rows of OHCscould be seen using a calbindin-antibody that labels hair cells (FIG.12a). Cellular death was studied in paraffin sections stained by theTUNEL-method that labels fragmented DNA. Further, when TUNEL-positivecells were found, DNA fragmentation was verified by using the DAPIcounterstain. TUNEL-labeled hair cells were not found in normal explants(n=5). In contrast, in explants (n=15) exposed to 100 μM neomycin for 6,12, 24 and 48 hr, TUNEL-positive hair cells were found, most of them atthe first 2 time points studied (FIG. 12b,c). The majority of labeledhair cells were located in the basal cochlear turn where hair cells aremost sensitive to ototoxic antibiotics. TUNEL-positive hair cell nucleiwere found within the epithelium (FIG. 12b,c), and, in addition, haircells that had been extruded from the epithelium in the cultures showednuclear fragmentation.

In cochlear explants (n=10) exposed to 100 μM neomycin for 6 and 12 hr,phospho-JNK (FIG. 12d) and phospho-c-Jun (FIG. 12e) immunoreactive haircells were found in the lesioned regions, in the basal and upper middlecochlear turns. Only the nuclei of hair cells were stained by thesephospho-specific antibodies. Hair cells situated in the apical,nonlesioned areas did not show phospho-JNK or phospho-c-Junimmunolabeling, suggesting that the JNK pathway is involved in hair-cellstress responses. Formula II has been shown to attenuate neuronalapoptosis by blocking activation of JNK (Maroney et al., 1998). Whencochlear explants (n=5) were coincubated with 500 nM Formula II andneomycin, both TUNEL-labeling and induction of JNK and c-Junphosphorylation (FIG. 12b,c) were prevented in hair cells. These dataindicate that Formula II is effective in blocking JNK activation instressed hair cells.

EXAMPLE 8 Preparation of6H,12H,13H-Indeno[2,3-a]pyrrolo[3,4-c]carbazole-5,7(5H, 7H)-dione

Step A: Preparation of 2-(2(2-Hydroxy)indanyl)indole

n-BuLi (107.5 mmol, 43 mL of 2.5M solution in hexanes) was addeddropwise (15 min) to a solution (12.0 g, 102.4 mmol) of indole in dryTHF (400 μL) at −78° C. (nitrogen atmosphere). The solution was stirredfor 30 min, then CO₂ (g) was passed through the solution for 10 min. Theclear solution was allowed to warm to ambient temperature, then it wasconcentrated to half the original volume at reduced pressure. THF (200mL) was added and the solution re-cooled to −78° C. At this point,t-BuLi (102 mmol, 60 mL of 1.7M solution in hexanes) was added dropwise(45 min). The resulting yellow solution was allowed to stir for 2 h at−78° C. Next, 2-Indanone (15.0 g, 112.6 mmol) in THF (100 mL) was addeddropwise (30 min) and the mixture stirred for 1 hour. The reaction wasquenched by addition of water (5 mL); the resulting mixture was pouredinto saturated NH₄Cl solution (250 mL), and then extracted with ether(1×mL). The ether layer was washed with 100 mL saturated NH₄CI, dried(MgSO₄), and concentrated at reduced pressure to give an oily product.The product (V) was recrystallized from Et₂O-hexane to give 10.5 g of atan powder with a m.p. of 244°-245° C. The following NMR data wereobtained: ¹H NMR (CDCl₃): δ2.4 (bs, 1H), 3.3 (d, 2H), 3.6 (d, 2H), 6.4(s, 1H), 7.1-7.4 (m, 7H), 7.6 (d, 1H), 8.6 (bs, 1H). Anal. calc.C₁₇H₁₅NO; C, 81.90; H, 6.06; N, 5.62. Found C, 82.16; H, 6.03; N, 5.58.

The mother liquor was concentrated to yield an oily product. Columnchromatography (silica gel, EtOAc:hexane 1.2) yielded an additional 2.1g of product for a total yield of 12.6 g (49%).

Step B: Preparation of 2-(2-Indenyl)indole

To a stirred solution of 2-(2-(2-hydroxy)indanyl)indole (4.0 g, 16.1mmol) in acetone (30 mL) was added 2N HCl (10 mL). The mixture wasstirred at ambient temperature for 1 hour. About 20 mL of water wereadded and the precipitate collected by filtration. The filtrate waswashed well with water and dried to give 3.6 g (98%) of a white solidproduct with a m.p. of 273°-274° C. (MeOH). The following NMR data wereobtained: ¹H NMR (CDCl₃): 63.9 (s, 2H), 6.7 (s, 1H), 7.0-7.6 (m, 9H),8.3 (bs, 1H). Anal.calc. C₁₇H₁₃N, C, 88.28; H, 5.67; N, 6.06. Found C,88.11; H, 5.60; N, 5.98.

Step C: Preparation of 4c,7a,7b,12a-Tetrahydro-6H, 1 2H 13H-indeno[2,3-a]pyrrolo [3,4-c]carbazole-5,7-(5H, 7H)dione

A mixture of 2-(2-indenyl)indole (1.0 g, 4.3 mmol) and maleimide (525mg, 5.41 mmol) in a 10 cm sealed reaction vial was heated at 180°-190°C. for 30 min. After cooling the reaction to ambient temperature, MeOH(5 mL) was added. The product was collected to give 880 mg (62%) of awhite solid product with a m.p. of 254°-255° C. (MeOH). The followingNMR data were obtained: ¹H NMR (DMSO-d₆, 300 MHz): 63.1-3.4 (m, 2H), 3.8(m, 2H), 3.95 (t, 1H), 4.35 (d, 1H), 6.9-7.4 (m, 7H), 7.75 (d, 1H),11.05 (s, 1H), 11.25 (s, 1H).

Step D: Preparation of6H,12H,13H-Indeno[2,3-a]pyrrolo[3,4-c]carbazole-5,7(5H, 7H)-dione

4c,7a,7b, 1 2a-Tetrahydro-6H, 12H, I 3H-indeno[2,3-a]pyrrolo[3,4-c]carbazole-5,7-(5H, 7H)dione (800 mg, 2.44 mmol) was dissolved intoluene (60 mL). Solid 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (1.4 g,6.1 mmol) was added to the toluene solution in one portion.

The solution was maintained at 60°-65° C. for 6 hours. After cooling onan ice bath, the solid product was collected by filtration, resuspendedin MeOH (20 mL) and collected by filtration.

The product was recrystallized from acetone-MeOH to yield 710 mg (90%)of a yellow solid product with a m.p. greater than 330° C. The followingNMR data were obtained: ¹H NMR (DMSO-d₆, 300 MHz): δ4.3 (s, 2H), 7.35(t, 1H), 7.45-7.65 (m, 4H), 7.75 (d, 1H), 8.95 (d, 1H), 9.1 (d, 1H),11.15 (s, 1H), 12.3 (s, 1H). MS(FAB): m/e 325 (m+1)⁺. Anal. calc. forC₂₁H₁₂N₂O₂. 0.75 H₂O: C, 74.65; H, 4.03; N, 8.29. Found; C, 74.40; H,3.75; N, 8.26.

EXAMPLE 9 Preparation of13-(2-Hydroxyethyl):5H;6H,12H,13H-indeno[2,3-a]pyrrolo [3,4-c]carbazole-7(7H)one

Step A: Preparation of 5H,6H,12H,13H-Indeno[2,3-a]pyrrolo[3,4-c]carbazole-7(7H)one

A stirred suspension of Zn dust (5 g) and mercuric chloride (1 g) wasmade in 10 mL water. Concentrated hydrochloric acid (2 mL) was addeddropwise. After 10 min, the aqueous layer was decanted and removed. Thezinc amalgam obtained was first washed with water, then repeatedly withEtOH. The zinc amalgam was suspended in EtOH (75 mL). Next, solid6H,12H,13H-Indeno[2,3-a]pyrrolo[3,4-c]carbazole-5,7(5H,7H)-dione (500mg, 1.5 mmol) was added in one portion. HCl(g) was passed through as themixture was maintained at reflux for 2 hours. After cooling to ambienttemperature, the solution was concentrated at reduced pressure to yieldan oily product. THF-EtOAc (200 mL, 1:1) was added to the oily productand the mixture was extracted with a saturated NaHCO₃ solution (3×100mL), saturated NaCI solution (3×100 mL) and the resulting solution dried(MgSO₄). The drying agent was removed, and the solvent was concentratedat reduced pressure to give a crude solid. Purification by columnchromatography (silica gel, 95:5, EtOAc:MeOH) yielded 240 mg (50%) of a1:4 mixture of 5H,6H,12H,13H-indeno[2,3-a]pyrrolo[3,4-c]carbazole-7(7H)one and6H,7H,12H,13H-indeno[2,3-a]pyrrolo[3,4-c]carbazole-5(5H)one. Thefollowing NMR data were obtained: ¹H NMR (DMSO-d₆, 300 MHz): 64.15 (s,1.6H), 4.25 (s, 0.4H), 4.9 (s, 0.4H), 4.95 (s, 1.6H), 7.2-7.8 (m, 6H),8.0 (d, 1H), 8.6 (s, 0.8H), 8.8 (s, 0.2H), 9.2 (d, 0.2H), 9.4 (d, 0.8H),11.8 (s, 0.2H), 11.95 (s, 0.8H). MS(m/e 311 (m+l )⁺.

Step B: Preparation of 13-(2-Hydroxyethyl):5H;6H,

12H.1 3H-indeno[2,3-a]pyrrolo[3,4-c]carbazole-7(7H)one

5H,6H,12H,13H-indeno[2,3-a]pyrrolo[3,4-c]carbazole-7(7H)one (200 mg,0.65 mmol) was added to a stirred solution of NaH (25 mg of 60% oildispersion, 0.65 mmol) in dry DMF (10 mL) under a nitrogen atmosphere.The dark mixture was stirred at ambient temperature for 1 hour. Ethylbromoacetate (120 mg, 0.08 mL, 0.72) was added dropwise and the mixturewas stirred 12 hours. The resulting yellow solution was concentrated atreduced pressure to give a crude yellow solid. The product was dissolvedin dry THF (10 mL) and lithium aluminium hydride (1 mL of 1M solution inether) was added dropwise. The solution was stirred 6 hours at roomtemperature, then the reaction was quenched by the addition of H₂O (1mL). The mixture was filtered and concentrated at reduced pressure. THFwas added to the residue and the product was collected to give 30mg(17%) of 13-(2-Hydroxyethyl):5H;6H, 12H,13H-indeno[2,3-a]pyrrolo[3,4-c]carbazole-7(7H)one as a white solid. Themelting point was greater than 300° C. The following NMR data wereobtained: ¹H NMR (DMSO-d₆, 300 MHz): δ3.8-3.9 (b, 2H), 4.55 (s, 2H),4.77 (t, 2H), 4.9 (s, 2H), 5.0 (1H, D₂O exchange), 7.3-7.45 (m, 3H),7.5-7.57 (t, 1H), 7.67 (d, 1H, J=6 Hz), 7.5 (d, 1H, J=6 Hz), 8.0 (d, 1H,J=6 Hz); 8.57 (s, 1H), 9.5 (d, 1H, J=7 Hz). MS(FAB): m/e 355 (M+1)⁺.

EXAMPLE 10 Preparation of6H,12-Benzo[b]thieno[2,3-pyrrolo[3,4-c]carbazole-5,7(5H, 7H)dione

A solution of 2-(2-benzo[b]thienyl)indole, maleimide (120 mg, 1.2 mmol)and trifluoroacetic acid (1 mL) in dry toluene (75 mL) was stirred atreflux for 12 hours. The solution was cooled to ambient temperature andconcentrated at reduced pressure to yield a crude solid. The solid wasdissolved in glacial HOAc (40 mL), 5% Pd(OAc)₂ was added and the mixturemaintained at reflux for 12 hours. The solution was cooled to ambienttemperature, filtered through Celite™, then concentrated at reducedpressure. MeOH was added to the residue and the product collected (80mg, 23%). The product was further purified by column chromatography(EtOAc:Hexane 2:1 R_(f)=0.5) to give6H,12-Benzo[b]thieno[2,3-pyrrolo[3,4-c]carbazole-5,7(5H, 7H)dione. Themelting point was greater than 300° C. The following NMR data wereobtained: ¹H NMR (DMSO-d₆, 300 MHz) δ7.4 (t, 1H), 7.55-7.75 (m, 4H),8.25 (m, 1H), 9.05 (d, 1H), 9.8 (m, 1H), 11.4 (s, 1H), 12.8 (s, 1H).MS(FAB): m/e=343 (M+1)⁺. Anal. calc. for: C₂₀H₁₀N₂OS. 0.5 H₂O; C, 67.49;H, 3.26; N, 7.87. Found: C, 67.50; H, 3.07; N, 7.51.

EXAMPLE 11 Preparation of the Following Fused Pyrrolocarbazole

Preparation of 2:

The diene 1 (50 mg, 0.182 mmol), 7.5 mg (0.018 mmol) of ytterbiumbromide and 68 mg (3 eq) of ethyl cyanoacrylate in 1 mL of toluene washeated under reflux for 3.5 hr. The product precipitated when themixture was allowed to cool to room temperatture. The solid wasfiltered, washed with toluene to give 48 mg (66% theory) of the product2 as off white solid. It was shown to be homogeneous by hplc. ¹H NMR(DMSO-d6, 300 MHz): d 11.2 (s, 1H), 7.55 (bq, 1H), 7.35 (d, 1H), 7.2(bq, 1H), 7.1 (t, 1H), 7.05 (t, 1H), 6.7 (bq, 1H), 6.55 (s, 1H), 3.7 (s,3H), etc.

Preparation of 3:

To 60 g, (0.150 mol) of the diels alder adduct 2 suspended in 1.5 L oftoluene was added 71.4 g (0.315 mol, 5% excess) of DDQ at roomtemperature with vigorous stirring. The temperature of the reactionmixture gradually rose to 33° C. over 1 hour period before returning toroom temperature. The solid was collected on a filter, washed thoroughlywith toluene and air dried. It was then dispersed in 2 L of water withvigorous stirring, and 80 g (theory 52.8 g) of sdium bicarbonate wasadded portion-wise. After stirring for 2 to 3 hours, the mixture wasfiltered and the solid was washed thoroughly with water until thewashings were neutral. The crude product 3 weighed 61.5 g (96% purity byhplc). 1H NMR (DMSO-d6, 300 MHz): d 12.1 (s, 1H), 8.45 ( d, 1H), 7.60(m, 2H), 7.25-7.4 (m, 2H), 7.0 (s, 1H), 6.9 (s, 1H), 4.35 (q, 2H), 3.8(s, 3H), 3.15 (m, 2H), 2.9 (m,2H), 1.25 (t, 3H). The crude product wasused for the next step without further purification.

Preparation of 4:

A mixture of 50 g (0.126 mol) of the cyanoester 3, 120 g (0.56 mol, 4.43eq) of 2-bromoethylbenzyl ether and 250 mL of 10N sodium hyroxide in1450 mL of acetone was heated under reflux overnight. Most of theacetone was removed under reduced pressure, 500 mL of water and 1250 mLof hexane were added and the mixture was stirre vigorously for 0.5 hr.The resulting solid was filtered, washed thouroughly with water untilthe washing became neutral. The solid was dried under vacuum, thenwashed with hexane to give 60 g of the product 4 (89.5% theory, 95%purity by hplc). 1H NMR (DMSO-d6, 300 MHz): d 8.55 (d, 1H), 7.85 (d,1H), 7.15 (t, 1H), 7.4 (t, 1H), 7.25 (d, 1H), 7.15 (m, 3H), 7.0 (s, 1H),6.9 (m, 3H), 4.9 (bs, 2H), 4.30 (m, 4H), 3.8 (m, 5H), 3.45 (t, 2H), 2.75(t, 2H), 1.2 (t, 3H).

Preparation of 5:

A solution of 59.8 g (0.113 mol) of 4 in 1 L of DMF containing 100 mL ofmethanol was hydrogenated over 100 g of Raney nickel at 55 psi on a Parrapparatus. The catalyst was removed by filtration and the filtrate wasconcentrated under reduced pressure and the resulting semi-solid wastriturated with 1.8 L of ether overnight to give 53.6 g of the lactam(93% theory) which was shown to contain 4% of further reduced product(debenzylated, 5104), 92% of desired product 5 and 4% impurity by hplc.1H NMR (DMSO-d6, 300 MHz): d 8.45 (s, 1H), 7.95 (d, 1H), 7.85 (d, 1H),7.7 (d, 1H), 7.50 (t, 1H), 7.3 (t, 1H), 7.15 (bq, 3H), 7.05 (bq, 2H),6.85 (s, 1H), 6.80 (d, 1H), etc. The crude material was used for thesubsequent reaction without further purification.

Preparation of the title compound

A solution of 43.6 g (89.2 mmol) of the lactam 5 in 1 L of DMFcontaining 5 drops of 12M hydrochloric acid was hydrogenated over 2.2 gof palladium hydroxide at 50 psi. The catalyst was removed by filtrationthrough a bed of Celite, and the filtrate was concentrated under reducedpressure and the resulting semisolid was triturated with 2 L of etherovernight to give CEP-5104 as a pale yellowish solid, 36 g (100% theory,97% purity by hplc. 1H NMR (DMSO-d6, 300 MHz): d 8.4 (s, 1H), 7.95 (d,1H), 7.85 (d, 1H), 7.7 (d, 1H), 7.50 (t, 1H), 7.3 (t, 1H), 6.9 (s, 1H),6.8 (d, 1H), 5.0 (bs, 1H), 4.8 (s, 2H), 4.65 (bt, 2H), 3.8 (bs, 5H), 3.3(bt, 2H), 2.75 (bt, 2H). MS (ES+) m/e 399 (M+1).

EXAMPLE 12

The following fused pyrrolocarbazoles were prepared using the methodsdescribed herein.

EXAMPLE 12A

EXAMPLE 12B

EXAMPLE 12C

EXAMPLE 12D

EXAMPLE 13 Preparation of13-(3-hydroxypropyl)-3-(pyridyl-2-thiomethyl)-6H,12H-indeno[2,3-a]pyrrolo[3,4-c]carbazole-7(7H)one

PREPARATION OF 2:

To a suspension of 1 (8.0 g, 0.258 mols) in acetonitrile (300 mL) atroom temperature under nitrogen was added ethyl acrylate (4.19 mL, 0.387mols) followed by 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) (1.93 mL,0.013 mols). After addition of DBU, the reaction changed colors fromorange to green. The reaction mixture was heated to reflux overnight.The mixture remained heterogeneous throughout the course of the reactionand became dark in color. A small aliquot was removed after 18 h and thesolid was collected by filtration. 1H NMR of the sample showed nostarting material remaining. The reaction mixture was cooled to roomtemperature and the solid was collected by filtration. The solid waswashed several times with cold acetonitrile and dried in vacuo at 55° C.to yield a light orange solid (5.4 g, 78% yield). 1H NMR (DMSO-d6, 300MHz): d 9.72 (t, 3H, J=6.8), 2.87 (m, 2H), 3.89 (q, 2H, J=6.8), 4.49 (s,2H), 4.88 (s, 2H), 4.92 (m, 2H), 7.29-7.48 (m, 3H), 7.50-7.73 (m, 3H),7.96 (d, 1H, J=7.33), 8.56 (s, 1H), 9.47 (d, 1H, J=7.33).

PREPARATION OF 3:

To a suspension of 2 (5.62 g, 0.0137 mols) in benzene (300 mL) andN-methylpyrrolidine (NMP) (60 mL) at room temperature under nitrogen wasadded p-toluenesulfonic acid monohydrate (2.48 g, 0.013 mols) and4,4′-dimethoxybenzhydrol (3.19 g, 0.013 mols). The contents of the flaskwere heated to reflux for 8 h. After 45 min., the initiallyheterogeneous reaction mixture became homogeneous. The reaction mixturewas cooled to room temperature, diluted with ethyl acetate (300 mL) andwashed with a saturated bicarbonate solution, water, and brine. Theorganic layer was dried over magnesium sulfate, filtered andconcentrated in vacuo to an orange solid (8.31 g, 95% yield). 1H NMR(CDCl3, 300 MHz): d 1.18 (t, 3H, J=7.1), 2.84 (m, 2H), 3.80 (6H, s),4.12 (q, 2H, J=7.1), 4.38 (s, 2H), 4.72 (2H, s), 4.94 (m, 2H), 6.90 (d,4H, J=8.5), 6.955 (s, 1H), 7.26 (d, 4H, J=8.5), 7.34-7.49 (m, 5H), 7.61(d, 1H, J=7.4), 7.69 (d, 1H, J=7.7), 9.65 (d, 1H, J=7.8).

PREPARATION OF 4:

To a stirred solution of 3 (7.8 g, 0.0122 mols) in THF (480 mL) andmethanol (93 mL) was added lithium borohydride (18.9 mL of a 2.0M soln,0.0379 mols) dropwise. The reaction mixture was initially homogeneous,however, as the reaction proceeded, the mixture became heterogeneous.When all of the starting material had been consumed, the reactionmixture was cooled in an ice bath and carefully quenched with 2N HCl (60mL). The reaction mixture became homogeneous and light orange in color.Water (750 mL) was added to the mixture and a milky white precipitateformed. The precipitate was collected by filtration and dried in vacuoto give a fluffy white solid (7.2 g, 99% yield). 1H NMR (DMSO-d6, 300MHz): d 1.93 (m, 2H), 3.66 (m, 2H), 3.71 (s, 6H), 4.55 (s, 2H), 4.73 (m,2H), 4.79 (s, 2H), 6.70 (s, 1H), 6.93 (d, 4H, J=8.44), 7.22 (d, 4H,J=8.4), 7.26 (m, 1H), 7.34-7.46 (m, 2H), 7.49 (m, 1H), 7.65 (d, 1H,J=7.01), 7.70 (d, 1H, J=8.26), 7.86 (d, 1H, J=7.82), 9.49 (d, 1H,J=7.49).

PREPARATION OF 5:

To a suspension of 4 (2.02 g, 0.0034 mols) in THF (131 mL) at roomtemperature under nitrogen was added N-bromosuccinimide (0.63 g, 0.0036mols) in one portion. The reaction mixture stirred at room temperatureovernight. The reaction solvent was removed in vacuo leaving a paleyellow solid. The solid was triturated with cold methanol and collectedby filtration. The solid was dried in vacuo to give a pale yellow solid(1.98, 87% yield). 1H NMR (DMSO-d6, 300 MHz): d 1.91 (m, 2H), 3.44 (m,2H), 3.72 (s, 6H), 4.53 (s, 2H), 474 (m, 2H), 4.87 (s, 2H), 6.71 (s,1H), 6.93 (d, 4H, J=8.14), 7.25 (d, 4H, J=8.1), 7.37 (m, 2H), 7.59-7.69(m, 3H), 8.08 (s, 1H), 9.50 (d, 1H, J=7.01).

PREPARATION OF 6:

In a Schlenk tube was placed 5 (0.79 g, 0.0017 mols) in methoxyethanol(25 mL) followed by sodium acetate (0.57 g, 0.00702 mols) anddichlorobis(triphenylphosphine)-palladium(II) (0.082 g, 0.000117 mols).The tube was evacuated and filled with carbon monoxide. The reactionmixture was heated in the sealed tube at 155° C. in an oil bath for 3 h.The reaction was cooled to room temperature and additional carbonmonoxide was added. The mixture was reheated to 150° C. for another 3 h.Additional CO and PdCl2(PPh3)2 were added and the mixture heated for 4h. The reaction mixture was diluted with methylene chloride and flushedthrough a pad of celite. The filtrate was concentrated in vacuo to aresidue which was dissolved in ethyl acetate and washed with water. Theorganic layer was dried over magnesium sulfate, filtered, andconcentrated in vacuo to a solid which was triturated with ethyl etherand collected by filtration to yield a light orange solid (0.7 g, 85%yield). 1H NMR (CDCl3, 300 MHz): d 2.14 (m, 2H), 3.44 (s, 3H), 3.67-3.78(m, 4H), 3.81 (s, 6H), 4.44 (s, 2H), 4.51 (m, 2H), 4.81 (m, 4H), 6.91(d, 4H, J=8.53), 6.98 (s, 1H), 7.28 ( d, 4H, 8.6), 7.34-7.7.61 (m, 4H),8.21 (d, 1H, J=8.32), 8.42 (s, 1H), 9.67 (d, 1H, J=7.61).

PREPARATION OF 7:

To a solution of 6 (0.96 g, 0.00138 mols) in CH2Cl2 (30 mL) at 0° C.under nitrogen was added thioanisole (3.2 mL, 0.110 mols) followed bytrifluoroacetic acid (TFA)(8.5 mL, 0.0276 mols). Upon addition of TFA,the reaction mixture turned red in color. The mixture stirred at 0° C.for 1 h and was warmed to room temperature overnight. The reactionsolvent was removed in vacuo leaving a dark red oil. Ethyl ether wasadded to the oil and the reaction mixture turned yellow in color and atan solid precipitated out of solution. The solid was collected byfiltration (0.6 g, 92% yield). 1H NMR (DMSO-d6, 300 MHz): d 2.29 (m,2H), 3.3 (m, 2H), 3.73 (m, 2H), 4.45 (m, 2H), 4.54 (m, 3H), 4.82 (m,2H), 4.99 (s, 2H), 7.40 (m, 2H), 7.58 (d, 1H), 7.85 (d, 1H), 8.13 (d,1H), 8.52 (s, 1H), 8.6 (s, 1H), 9.49 (d, 1H).

PREPARATION OF 8:

To a stirred suspension of 7 (4.4 g, 0.00935 mols) in CHCl2 (220 mL) at0° C. under nitrogen was added DIBAL-H slowly dropwise. The reactiongradually became homogeneious. The orange-colored reaction mixturestirred at 0° C. for 1 h then was warmed to room temperature and wasstirred for 6 h. The mixture was cooled to 0° C. in an ice bath andwater (50 mL) was added extremely slowly initially. Vigorous evolutionof gas was observed. An aqueous solution of NaOH (1M, 300 mL) was addedand the reaction mixture stirred at room temperature for 1 h. Aprecipitate formed and was collected by filtration to yield a tan solid(3.6 g, 96%). 1H NMR (DMSO-d6, 300 MHz): d 1.92 (m, 2H), 3.46 (m, 2H),4.50 (s, 2H), 4.65 (s, 2H), 4.71 (m, 2H), 4.88 (s, 2H), 7.32-7.39 (m,2H), 7.47 (d, 1H, J=8.34), 7.65 (m, 2H), 7.89 (s, 1H), 8.53 (s, 1H),9.46 (d, 1H, J=7.44).

PREPARATION OF 9:

To a suspension of 8 (105.6 mg, 0.265 mmols) in dioxane (3 mL) was added2-mercaptopyridine (73.7 mg, 0.663 mmols) and camphorsulfonic acid(184.7 mg, 0.795 mmols). The suspension was heated in a sealed tube at80° C. for 5 h during which time the reaction mixture became ahomogeneous solution. The reaction mixture was cooled to roomtemperature overnight. Thin layer chromatography (100% ethyl acetate)showed a small amount of starting material present so the mixture washeated for an additional 6 h at 80° C. After cooling to roomtemperature, the reaction mixture was triturated with ethyl ether. Asolid precipitated out of solution and was collected by filtration. Thesolid was suspended in ethyl acetate and washed with a saturated sodiumbicarbonate solution to remove the camphorsulfonic acid. The organiclayer was dried over sodium sulfate, filtered and concentrated in vacuoto a light orange solid. The solid was triturated with ethyl ether andcollected by filtration to yield pure product (67 mg, 51% yield). MS(ESI): m/e 514 (M+Na)⁺, 1H NMR (DMSO-d6, 300 MHz): d 1.014 (m, 2H), 3.45(m, 2H), 4.51 (s, 2H), 4.60 (s, 2H), 4.72 (m, 3H), 4.85 (s, 2H), 7.11(m, 1H), 7.30-7.41 (m, 3H), 7.54-7.67 (m, 4H), 8.02 (s, 1H), 8.48 (d,1H, J=3.97), 8.55 (s, 1H), 9.46 (d, 1H, J=7.36).

EXAMPLE 14

The following fused pyrrolocarbazoles were prepared using the methodsdescribed herein.

EXAMPLE 14A

EXAMPLE 14B

EXAMPLE 15 Protective effect of the different compounds of Formula I andthe compound of Formula II against neomycin in neonatal rat cochlearexplants

The compounds of Formula I and the compound of Formula II attenuatedneomycin-induced cochlear sensory hair cell loss in vitro. The effect ofthe compounds of Formula I and the compound of Formula II onneomycin-induced hair cell degeneration was determined in organotypiccultures of the neonatal organ of Corti.

Cochlear cultures.

The basal half of cochleas containing the basal and middle turns weredissected from postnatal day 2 Wistar rats. The cultures were maintainedon Nuclepore filters (pore size 0.1 mm; Pleasanton, Calif.) placed on ametal grid in F12 medium (Life Technologies, Gaithersburg, Md.)containing 15% fetal bovine serum (Life Technologies). After a2-hour-long stabilization period, explants were exposed to 100 μMneomycin sulfate (Sigma, St Louis, Mo.) for 48 hr. The compounds ofFormula I and the compound of Formula I were added at 500 nM at the timeof initiation of the cultures and every 12 hr thereafter. Neomycin plusvehicle was used as a control.

Hair cell counts in cochlear cultures.

Explants were fixed with 4% paraformaldehyde/0.5% glutaraldehyde inphosphate buffered saline (PBS), pH 7.4, for 30 min and dissected forsurface preparations. They were stained with a 1:100 dilution ofrhodamine-phalloidin in PBS containing 0.25% Triton X-100 overnight at4° C. and mounted in Vectashield (Vector). Outer hair cell (OHC) numberswere quantified with a Zeiss Axiovert 100/135 epifluorescent microscope(Germany) connected to a Bio-Rad MRC-1024 confocal laser scanning system(Richmond, Calif.). Hair cells were characterized as missing if nostereocilia and cuticular plate were observed. Numbers of OHCs wereevaluated using a 40x objective lens and an ocular grid. Several fieldsfilled by 30 OHCs in each of the 3 rows (when all of them were present)were studied from each explant. Basal and middle coils were analyzedseparately.

Results

When the cultures were treated with 100 μM neomycin, many sensory haircells were lost within 48 hours. 100μM neomycin caused the loss of mostbasally located sensory hair cells, as detected in surface preparationsof the cultures using F-actin (phalloidin) as a sensory hair cellmarker. When 500 nM of the compounds of Formula I was added to thecultures together with neomycin, different levels of sensory hair cellprotection was achieved. Table 6 shows the protection against loss ofsensory hair cell degeneration by coincubation with the compounds ofFormula I and the compound of Formula II. Datapoints marked with “**”indicate results which are thought to be indicative of artifacts.

TABLE 6 Compounds of Formula I and Formula II in Cochlear CulturesCompound # fields total HCs missing HCs % preserved Example 8 11 13201040 21 Example 8 9 1080 850 21 Example 9 15 1560 1060 32 Example 9 101200 890 26 Example 9 9 1080 240  78** Example 9 8 960 840 13 Example 108 960 115 88 Example 10 10 1200 20 98 Example 10 10 1200 120 98 Example10 7 840 20 98 Example 10 7 840 200 76 Example 11 6 720 585 19 Example11 8 960 420 56 Example 11 8 960 620 35 Example 11 13 1560 845 46Example 12A 6 720 500 25 Example 12A 7 840 500 60 Example 12A 8 960 70035 Example 12A 10 1200 1100 10 Example 12A 5 600 120 20 Example 12B 6720 20 97 Example 12B 18 2160 560  74** Example 12B 4 480 20 95 Example12C 8 960 720 25 Example 12C 10 1200 600 50 Example 12C 8 960 860 10Example 12D 10 1200 1000 17 Example 12D 7 840 725 12 Example 12D 8 960620 35 Example 13 4 480 80 83 Example 13 6 720 270 62 Example 13 10 1200570 50 Example 14A 11 1320 910 31 Example 14A 8 960 840 12 Example 14A 8960 900  6 Example 14B 9 1080 520 50 Example 14B 8 960 830 15 Example14B 9 1080 1005  5 Formula II 7 840 70 92 Formula II 8 960 165 83Fonnula II 10 1200 60 95 Formula II 4 480 15 97 NEO 11 1320 1270  4 NEO7 840 680 19 NEO 9 1080 1040  4 NEO 12 1440 1210 16

The disclosures of each patent, patent application and publication citedor described in this document are hereby incorporated by referenceherein in their entirety.

Various modifications of the invention, in addition to those describedherein, will be apparent to those skilled in the art from the foregoingdescription. Such modifications are also intended to fall within thescope of the appended claims.

What is claimed is:
 1. A method for preventing hearing loss in asubject, said method comprising: administering to said subject aneffective amount of a fused pyrrolocarbazole of Formula I having theformula:

or a stereoisomer or pharmaceutically acceptable salt form thereof,wherein: ring D is selected from phenyl and cyclohexene with double bonda-b; ring B and ring F are independently selected from: (a) a 6-memberedcarbocyclic ring in which from 1 to 3 carbon atoms may be replaced byheteroatoms; (b) a 5-membered carbocyclic ring; and (c) a 5-memberedcarbocyclic ring in which either: (1) one carbon atom is replaced withan oxygen, nitrogen, or sulfur atom; (2) two carbon atoms are replacedwith a sulfur and a nitrogen atom, an oxygen and a nitrogen atom, or twonitrogen atoms; or (3) three carbon atoms are replaced with threenitrogen atoms, one oxygen and two nitrogen atoms, or one sulfur and twonitrogen atoms; G-X-W is selected from: (a) (Z¹Z²)C-N(R¹)-C(Z¹Z²); (b)CH(R¹)-C(═O)-N(R¹); and (c) N(R¹)-C(═O)-CH(R¹); Z¹ and Z², at eachoccurrence, are independently selected from H, H; H, OR; H, SR; H,N(R)₂; and a group wherein Z¹ and Z² together form a moiety selectedfrom ═O, ═S, and ═NR; with the proviso that at least one of the pairs Z¹and Z² forms ═O; R is independently selected from H, optionallysubstituted alkyl, OH, alkoxy, OC(═O)R^(1a), OC(═O)NR^(1c)R^(1d),O(CH₂)pNR^(1c)R^(1d), O(CH₂)_(p)OR^(1b), optionally substitutedarylalkyl and optionally substituted heteroarylalkyl; R¹ isindependently selected from: (a) H, optionally substituted alkyl,optionally substituted aryl, optionally substituted arylalkyl,optionally substituted heteroaryl and optionally substitutedheteroarylalkyl; (b) C(═O)R^(1a); (c) OR^(1b); (d) C(═O)NHR^(1b),NR^(1c)R^(1d), (CH₂)pNR^(1c)R^(1d), (CH₂)_(p)OR^(1b), O(CH₂)_(p)OR^(1b)and O(CH₂)_(p)NR^(1c)R^(1d); R^(1a) is independently selected fromoptionally substituted alkyl, optionally substituted aryl and optionallysubstituted heteroaryl; R^(1b) is independently selected from H andoptionally substituted alkyl; R^(1c) and R^(1d) are each independentlyselected from H, optionally substituted alkyl and a linking group of theformula (CH₂)₂—X¹—(CH₂)₂; X¹ is independently selected from O, S andCH₂; Q is selected from NR², O, S, NR²², CHR²³, X⁴CH(R²³), CH(R²³)X⁴,wherein X⁴ is selected from O, S, CH₂, NR²² and NR²; R² is selected fromH, SO₂R^(2a), CO₂R^(2a), C(═O)R^(2a), C(═O)NR²CR^(2d), optionallysubstituted alkyl, optionally substituted alkenyl and optionallysubstituted alkynyl, wherein said optional substituents are one to aboutthree R⁵ groups; R^(2a) is independently selected from optionallysubstituted alkyl, optionally substituted aryl, optionally substitutedcarbocyclyl, optionally substituted heterocyclyl, OR^(2b), CONH₂,NR^(2c)R^(2d), (CH₂)_(p)NR^(2c)R^(2d) and O(CH₂)_(p)NR^(2c)R^(2d);R^(2b) is selected from H and optionally substituted alkyl; R^(2c) andR^(2d) are each independently selected from H and optionally substitutedalkyl, or together form a linking group of the formula (CH₂)₂—X′—(CH₂)₂;R³ and R⁴ are each independently selected from: (a) H, aryl, heteroaryl,F, Cl, Br, I, CN, CF₃, NO₂, OH, OR⁹, O(CH₂)_(p)NR¹¹R¹², OC(═O)R⁹,OC(═O)NR¹¹R¹², O(CH₂)_(p)OR¹¹, CH₂OR¹⁰, NR¹¹R¹², NR¹⁰S(═O)₂R⁹ andNR¹⁰C(═O)R⁹; (b) CH₂OR¹⁴; (c) NR¹⁰C(═O)NR¹¹R¹², CO₂R¹⁰, C(═O)R⁹,C(═O)NR¹¹R¹², CH═NOR¹⁰, CH═NR¹⁰, (CH₂)_(p)NR¹¹R¹², (CH₂)_(p)NHR¹⁴ andCH═NNR¹¹R¹²; (d) S(O)_(y)R⁹, (CH₂)_(p)S(O)_(y)R⁹ and CH₂S(O)_(y)R¹⁴; (e)optionally substituted alkyl, optionally substituted alkenyl andoptionally substituted alkynyl, wherein said optional substituents areone to about three R⁵ groups; R⁹ is selected from alkyl, (CH₂)_(r)aryland (CH₂)_(r)heteroaryl; R¹⁰ is selected from H, alkyl, (CH₂)_(r)aryland (CH₂)_(r)heteroaryl; R¹¹ and R¹² are independently selected from Hand optionally substituted alkyl, or together form a linking group ofthe formula (CH₂)₂—X¹—(CH₂)₂; R⁵ is selected from aryl, heteroaryl,arylalkoxy, heterocycloalkoxy, hydroxyalkoxy, alkyloxy-alkoxy,hydroxyalkylthio, alkoxy-alkylthio, F, Cl, Br, I, CN, NO₂, OH, OR⁹,X²(CH₂)_(p)NR¹¹R¹², X²(CH₂)_(p)C(═O)NR¹¹R¹², X²(CH₂)_(p)OC(═O)NR¹¹R¹²,X²(CH₂)_(p)CO₂R⁹, X²(CH₂)_(p)S(O)_(y)R⁹, X²(CH₂)_(p)NR¹⁰C(═O)NR¹¹R¹²,OC(═O)R⁹, OC(═O)NHR¹⁰, O-tetrahydropyranyl, NR¹¹R¹², NR¹⁰C(═O)R⁹,NR¹⁰CO₂R⁹, NR¹⁰C(═O)NR¹¹R¹², NHC(═NH)NH₂, NR¹⁰S(O)₂R⁹, S(O)_(y)R⁹,CO₂R¹⁰, C(═O)NR¹¹R¹², C(═O)R⁹, CH₂OR¹⁰, CH═NNR¹¹R¹², CH═NOR¹⁰, CH═NR⁹,CH═NNHCH(N═NH)NH₂, S(═O)₂NR¹¹R¹², P(═O)(OR¹⁰)₂, OR¹⁴, and amonosaccharide wherein each hydroxyl group of the monosaccharide isindependently either unsubstituted or is replaced by H, alkyl,alkylcarbonyloxy, or alkoxy; X² is O, S, or NR¹⁰; Y is selected from:(a) a direct bond; (b) optionally substituted CH₂, CH₂CH₂ or CH₂CH₂CH₂,wherein said optional substituents are one to about three R¹⁹ groups;and (c) CH═CH, CH(OH)—CH(OH), O, S, S(═O), S(═O)2, C(R¹⁸)₂, C═C(R¹⁹)₂,C(═O), C(═NOR²⁰), C(OR²⁰)R²⁰, C(═O)CH(R¹⁸), CH(R¹⁸)C(═O),C(═NOR²⁰)CH(R¹⁸), CHR²¹C(═NOR²⁰), C(═O)N(R²¹), N(R²¹)C(═O), CH₂Z, ZCH₂and CH₂ZCH₂, where Z is selected from C(R²¹)₂, O, S, CO₂R²⁰, C(═NOR²⁰)and N(R²⁰); R¹⁸ is independently selected from H, SO₂R^(18a),CO₂R^(18a), C(═O)R^(18a), C(═O)NR_(18c)CR^(18d), optionally substitutedalkyl, optionally substituted alkenyl, and optionally substitutedalkynyl; R^(18a) is independently selected from optionally substitutedalkyl, optionally substituted aryl, optionally substituted carbocyclyland optionally substituted heterocyclyl; R^(18a) and R^(18d) are eachindependently selected from H and optionally substituted alkyl, ortogether form a linking group of the formula (CH₂)₂—X′—(CH₂)₂; R¹⁹ isindependently selected from R²⁰, thioalkyl, halogen, optionallysubstituted alkyl, optionally substituted alkenyl and optionallysubstituted alkynyl; R²⁰ is independently selected from H, alkyl, OH,alkoxy, OC(═O)R^(18a), OC(═O)NR^(18c)R^(18d), OC(═S)NR^(18c)R^(18d),O(CH₂)_(p)NR^(18c)R^(18d), O(CH₂)_(p)OR²¹, optionally substitutedarylalkyl, optionally substituted heterocyclylalkyl and optionallysubstituted carbocyclyl; R²¹ is independently selected from H and alkyl;Q′ is selected from: (a) a direct bond; (b) NR⁶; (c) optionallysubstituted CH₂, CH₂CH₂ or CH₂CH₂CH₂; (d) CR²²R²⁴; and (e) CH═CH,CH(OH)CH(OH), O, S, S(═O), S(═O)₂, C(═O), C(═NOR¹¹), C(OR¹¹)(R¹²),C(═O)CH(R¹³), CH(R¹³)C(═O), C(R¹⁰)₂, C(═NOR¹¹)CH(R¹³), CH(R¹³)C(═NOR¹¹),CH₂Z′, Z′—CH₂ and CH₂Z′CH₂; Z′ is selected from C(R¹¹)(OR¹²), O, S,C(═O), C(═NOR¹¹) and NR¹¹; R⁶ is selected from H, SO₂R^(2a), CO₂R^(2a),C(═O)R^(2a), C(═O)NR^(1a), CR^(1d), optionally substituted alkyl,optionally substituted alkenyl, and optionally substituted alkynyl,wherein said optional substituents are one to about three R⁵ groups; oralternatively, when Q is NR² and Q′ is NR⁶ or C(R¹⁰)₂, R² and R⁶ or oneof R¹⁰ are joined together to form:

wherein R⁷ and R⁸ are each independently selected from H, OH, alkyl,alkoxy, optionally substituted arylalkyl, optionally substitutedheteroarylalkyl, (CH₂)_(p)OR¹⁰, (CH₂)_(p)OC(═O)NR¹¹R¹² and(CH₂)_(p)NR¹¹R¹²; or R⁷ and R⁸ together form a linking group of theformula CH₂—X³—CH₂; X³ is a bond, O, S, or NR¹⁰; J is selected from abond, O, CH═CH, S, C(═O), CH(OR¹⁰), N(R¹⁰), N(OR¹⁰), CH(NR¹¹R¹²),C(═O)N(R¹⁷), N(R¹⁷)C(═O), N(S(O)_(y)R⁹), N(S(O)_(y)NR¹¹R¹²), N(C(═O)R⁷),C(R¹⁵R¹⁶), N⁺(O⁻)(R¹⁰), CH(OH)CH(OH) and CH(O(C═O)R⁹)CH(OC(═O)R⁹); J′ isselected from O, S, N(R¹⁰), N⁺(O⁻)(R¹⁰), N(OR¹⁰) and CH₂; R¹³ isselected from alkyl, aryl and arylalkyl; R¹⁴ is the residue of an aminoacid after the hydroxyl group of the carboxyl group is removed; R¹⁵ andR¹⁶ are independently selected from H, OH, C(═O)R¹⁰, O(C═O)R⁹, alkyl-OH,alkoxy and CO₂R¹⁰; R¹⁷ is selected from H, alkyl, aryl and heteroaryl;R²² is

X⁵ and X⁶ are independently selected from O, N, S, CHR²⁶, C(OH)R²⁶,C(═O) and CH₂═C; X⁷ and X⁸ are independently selected from a bond, O, N,S, CHR²⁶, C(OH)R²⁶, C(═O) and CH₂═C; X⁹ and X¹⁰ are independentlyselected from a bond, O, N, S, C(═O) and CHR²⁶; X¹¹ is a bond oralkylene optionally substituted with NR¹¹R¹² or OR³⁰; R²³ is selectedfrom H, OR²⁷, SR²⁷, R²² and R²⁸; R²⁴ is selected from R, thioalkyl, andhalogen; R²⁵ is selected from R¹ and OC(═O)NR^(1c)R^(1d); R²⁶ isselected from H, optionally substituted alkyl and optionally substitutedalkoxy, wherein (1) ring G contains 0 to about 3 ring heteroatoms; (2)any two adjacent hydroxyl groups of ring G can be joined to form adioxolane ring; (3) any two adjacent ring carbon atoms of ring G can bejoined to form a fused aryl or heteroaryl ring; with the provisos that:(a) when X¹¹ is a bond, ring G can be heteroaryl; and (b) ring G: (i)contains at least one carbon atom that is saturated; (ii) does notcontain two adjacent ring O atoms; (iii) contains a maximum of two C(═O)groups; R²⁷ is selected from H and alkyl; R²⁸ is selected from aryl,arylalkyl, SO₂R²⁹, CO₂R²⁹, C(═O)R²⁹, optionally substituted alkyl,optionally substituted alkenyl and optionally substituted alkynyl; R²⁹is selected from alkyl, aryl and heteroaryl; R³⁰ is selected from H,alkyl, acyl and C(═O)NR¹¹R¹²; m is independently selected from 0, 1, and2; p is independently selected from 1, 2, 3, and 4; r is independentlyselected from 0, 1, and 2; y is independently selected from 0, 1 and 2;and z is selected from 0, 1, 2, 3 and 4; with the provisos that at leastone of Y and Q′ is a direct bond, when Y is a direct bond, Q′ is otherthan a direct bond, when Q′ is a direct bond, Y is other than a directbond, and when rings B and F are phenyl, G-X-W is CH₂NHC(═O), Y is adirect bond, Q is NR² and Q′ is NR⁶ where R⁶ is joined with R² to form

then R³ is other than CH₂SCH₂CH₃.
 2. A method according to claim 1wherein, Y is a direct bond; and Q is NR₂.
 3. A method according toclaim 2 wherein ring B and ring F of the fused pyrrolocarbazole arephenyl, G-X-W is selected from CH₂NR¹C(═O), C(═O)NR¹CH₂, andC(═O)NR¹C(═O), and Q′ is NR⁶.
 4. A method according to claim 3 whereinthe fused pyrrolocarbazole has the formula:


5. A method according to claim 4 wherein R³ and R⁴ of the fusedpyrrolocarbazole are selected from H, alkyl, Cl, Br, CH₂OH, CH₂SOCH₂CH₃,CH₂SO₂CH₂CH₃, NHCONHC₆H₅, CH₂SCH₂CH₃, CH₂S-phenyl, CH₂S-pyridyl, CHNHCO₂CH₃, CH₂₀C(═O)NHCH₂CH₃, N(CH₃)₂, CH═NNH, CH₂N(CH₃)₂, andCH₂OCH₂CH₃; R⁷ is selected from H and alkyl; and R¹⁵ and R¹⁶ areindependently selected from H, alkyl, OH, CH₂OH, alkoxy, and CO₂alkyl.6. A method according to claim 5 wherein the fused pyrrolocarbazole hasthe formula:


7. A method according to claim 2 wherein Q′ is CH₂, CH₂CH₂, S orCH(CH(CH₃)(OH)).
 8. A method according to claim 7 wherein ring B andring F of the fused pyrrolocarbazole are phenyl and G-X-W is selectedfrom CH₂NR¹C(═O), C(═O)NR¹CH₂.
 9. A method according to claim 8 whereinthe fused pyrrolocarbazole has the formula


10. A method according to claim 9 wherein R² is H, CH₂CH₂OH,CH₂CH₂NHC(═O)—C₆H₅-OH, CH₂CH₂CH₂OH, R³ and R⁴ of the fusedpyrrolocarbazole are elected from H, alkyl, Cl, Br, alkoxy, CH₂OH,CH₂SOCH₂CH₃, CH₂SO₂CH₂CH₃, NHCONHC₆H₅, CH₂SCH₂CH₃, CH₂S-phenyl,CH₂S-pyridyl, CHNHCO₂CH₃, CH₂OC(═O)NHCH₂CH₃, N(CH₃)₂, CH═NNH,CH₂N(CH₃)₂, and CH₂OCH₂CH₃; R⁷ is selected from H and alkyl; and R¹⁵ andR¹⁶ are independently selected from H, alkyl, OH, CH₂OH, alkoxy, andCO₂alkyl.
 11. A method according to claim 10 wherein the fusedpyrrolocarbazole has the formula:


12. A method according to claim 8 wherein the fused pyrrolocarbazole hasthe formula: